TGGE MAXI user manual

TGGE MAXI user manual
TGGE MAXI System
Instruction Manual
Ver. 05/12
Model
Order No.
TGGE MAXI System
TGGE MAXI System (international)
024-200
024-290
Please read these instructions carefully before using this
apparatus!
Biometra GmbH
Rudolf-Wissell-Str. 30
D-37079 Göttingen
Tel.: ++49 (0)551-50 68 6-0
Fax: ++49 (0)551-50 68 6-66
email: [email protected]
Internet: http://www.biometra.com
Service Department
Rudolf-Wissell-Strasse 14-16
D-37079 Göttingen
Tel.: ++49 (0)551-50 68 6-10 or -12
Fax: ++49 (0)551-50 68 6-11
email: [email protected]
This document describes the state at the time of publishing. It needs not necessarily agree
with future versions.
Subject to change!
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Instruction Manual TGGE MAXI 05/2012
Contents
1.
Introduction.................................................................................................................. 6
1.1
Principle of the method .......................................................................................... 6
1.2
Field of Applications ............................................................................................... 6
1.3
Special features ..................................................................................................... 7
1.3.1
Peltier powered linear temperature gradient ................................................... 7
1.3.2
Sensitivity ....................................................................................................... 8
1.3.3
Patented technology....................................................................................... 8
1.4
Technical specifications ......................................................................................... 9
1.5
Legal Notes...........................................................................................................10
1.5.1
Copyright.......................................................................................................10
1.5.2
Liability ..........................................................................................................10
1.5.3
Meaning of the Instructions ...........................................................................10
2.
Safety and Warning Notices ......................................................................................11
2.1
Definition of Symbols ............................................................................................11
2.2
3.
4.
Safety instructions / general remarks ....................................................................11
Installation ..................................................................................................................13
3.1
Content of delivery ................................................................................................13
3.2
Unpack and Check................................................................................................13
3.3
Installation Conditions ...........................................................................................13
3.4
Operation Voltage .................................................................................................14
3.5
Setup ....................................................................................................................14
Considerations for successful TGGE experiments ..................................................15
4.1
Adaptation of protocols from the TGGE “mini” system...........................................15
4.2
Design of TGGE experiments ...............................................................................16
4.3
Design of DNA fragment for TGGE .......................................................................16
4.3.1
Poland analysis .............................................................................................16
4.3.2
GC clamps ....................................................................................................18
4.3.3
Chemical clamp with Psoralen (Furo[3,2-g]coumarin, C11H6O3) ....................19
4.3.4
Use of SSCP primers ....................................................................................19
4.4
Find correct temperature gradient .........................................................................20
4.5
5.
6.
Parallel analysis of multiple samples .....................................................................20
Sample preparation ....................................................................................................22
5.1
Purity of samples ..................................................................................................22
5.2
Sample preparation for direct DNA analysis ..........................................................22
5.3
Denaturation / Renaturation for heteroduplex analysis of DNA..............................22
Operating ....................................................................................................................23
6.1
Casting of gels ......................................................................................................23
6.1.1
Assembly of the gel cassette .........................................................................23
6.1.2
Preparing gel solution for mutation analysis ..................................................25
Instruction Manual TGGE MAXI 05/2012
3
6.1.3
Gel preparation for analysis of mixed bacterial populations (genetic fingerprint)
26
6.1.4
Pouring gels ..................................................................................................27
6.2
Electrophoresis .....................................................................................................27
6.2.1
Electrophoresis conditions.............................................................................27
6.2.2
Setup electrophoresis unit .............................................................................28
6.2.3
Prior to assembly of the electrophoresis unit .................................................28
6.2.4
Gel setup for electrophoresis .........................................................................29
6.2.5
Perpendicular TGGE .....................................................................................30
6.2.6
Parallel TGGE ...............................................................................................31
6.2.7
How to identify the optimum temperature range from a perpendicular gel .....31
7.
Programming the TGGE controller ............................................................................33
7.1
General overview ..................................................................................................33
7.1.1
Create / edit program ....................................................................................33
7.1.2
Select program ..............................................................................................33
7.1.3
Name program ..............................................................................................34
7.1.4
Enter temperatures for the gradient block ......................................................34
7.1.5
Enter electrophoresis parameters..................................................................35
7.1.6
Start electrophoresis .....................................................................................36
7.1.7
Stop/pause electrophoresis ...........................................................................36
7.1.8
View temperatures of the gradient .................................................................37
7.2
Special functions ...................................................................................................37
7.2.1
Print programs...............................................................................................38
7.2.2
Select / de-select signal ................................................................................38
7.2.3
Select language ............................................................................................38
7.2.4
Set block type................................................................................................38
7.3
Electrophoresis protocols for mutation analysis.....................................................38
7.3.1
Electrophoresis protocol for perpendicular TGGE* ........................................38
7.3.2
Electrophoresis protocol for parallel TGGE* ..................................................39
7.4
Electrophoresis protocols for mixed population analysis .......................................39
7.4.1
Electrophoresis protocol for perpendicular TGGE* ........................................39
7.4.2
Electrophoresis protocol for parallel TGGE* ..................................................40
8.
Staining of gels ...........................................................................................................41
8.1
Silver staining .......................................................................................................41
8.1.1
Silver staining protocol ..................................................................................41
8.2
Ethidium bromide-staining .....................................................................................42
9.
8.3
Autoradiography....................................................................................................42
8.4
Elution of DNA from the TGGE gels ......................................................................42
Trouble shooting guide ..............................................................................................43
9.1
Optimization of parallel TGGE...............................................................................44
9.2
Optimization of TGGE for genetic fingerprinting of microorganisms (mixed
population analysis) ..........................................................................................................45
9.3
Optimisation of Heteroduplex analysis ..................................................................45
9.3.1
Principle of heteroduplex analysis .................................................................45
9.3.2
Evaluation of a heteroduplex analysis ...........................................................46
9.4
The TGGE Test kit ................................................................................................47
9.4.1
Perpendicular TGGE using the Biometra TGGE test kit ................................47
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Instruction Manual TGGE MAXI 05/2012
9.4.2
Parallel TGGE using the Biometra TGGE test kit ..........................................48
10.
Maintenance and repair ..........................................................................................49
10.1 Cleaning and Maintenance ...................................................................................49
10.2
Servicing and repair ..............................................................................................49
10.3
Replacement of Spare Parts .................................................................................49
11.
Accessories ............................................................................................................50
11.1 Consumables and spare parts ..............................................................................50
12.
Service .....................................................................................................................51
12.1 Instructions for return shipment .............................................................................51
13.
Equipment Decontamination Certificate ...............................................................52
14.
Note for the disposal of electric / electronic waste. .............................................54
15.
Declaration of Conformity ......................................................................................55
16.
Warranty ..................................................................................................................56
17.
Appendix .................................................................................................................57
17.1 References ...........................................................................................................57
17.2 Buffers ..................................................................................................................63
17.2.1
Running buffers: ............................................................................................63
17.2.2
Loading buffers: ............................................................................................63
17.3 Other buffers: ........................................................................................................64
18.
Subject index ..........................................................................................................65
Instruction Manual TGGE MAXI 05/2012
5
1. Introduction
Temperature Gradient Gel Electrophoresis is a powerful technique for the separation of
nucleic acids or proteins. The TGGE method, which is covered by patents, uses the
temperature dependent changes of conformation for separating molecules (for review see
Reference 1).
Since the introduction of the first commercial available TGGE apparatus in 1989,
temperature gradient gel electrophoresis has gained high interest in scientific and clinical
research laboratories due to the unprecedented resolution capability and easiness of
analysis. The range of scientific publications using the TGGE method is broad and covers all
disciplines which use molecular biology methods: e.g. Oncology 2-4, Virology 5,6,
Immunology 7,8, RNA Viroid Research 9-12, Prion Research 13, Population Analysis 14-15.
The TGGE method has also been used for quantitative analysis in industry 16-17 and for
conformational analysis of proteins 18-19.
1.1 Principle of the method
Conventional protein or nucleic acid electrophoresis separates molecules according to their
size or charge. TGGE adds a new parameter for separation, namely the melting behavior
of a molecule. The melting behavior is determined by primary sequence and secondary and
tertiary structure of the molecule and can be changed by external influences like
temperature, salt concentration, pH etc.
During electrophoresis the sample migrates along a temperature gradient. As the
temperature rises the molecules start to denature. Working with PCR fragments for example
electrophoresis starts with double stranded molecules. At a certain temperature the DNA
starts to melt, resulting in a fork-like structure (partial single strand, see Figure 1). In this
conformation the migration is slowed down compared to a completely double stranded DNA
fragment (of same size). Since the melting temperature strongly depends on the base
sequence, DNA fragments of same size but different sequence can be separated. This is
used in mutation detection where PCR fragments of identical size but different sequence are
separated. Thus TGGE not only separates molecules but gives additional information about
melting behavior and stability.
electrophoresis
cold
ds DNA
partial ss
ss DNA
warm
Figure 1: Different conformations of DNA during temperature gradient gel electrophoresis.
1.2 Field of Applications
The Biometra TGGE MAXI system separates molecules in a temperature gradient. Unlike
chemical gradients the peltier driven temperature gradient is controlled by a microprocessor
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Instruction Manual TGGE MAXI 05/2012
and thus is providing unmatched reproducibility. The most common application for TGGE is
mutation analysis of PCR fragments. The DNA molecules become separated in the
temperature gradient their melting behavior. With TGGE PCR fragments that only have
single base substitutions can be resolved. But, the TGGE system is not restricted to the
analysis of DNA fragments only. It can be used for versatile applications like:
• Mutation analysis
• Heteroduplex analysis
• DNA methylation studies (Imprinting)
• Differentiation of amplicon and competitor for quantitative DNA analyses
• Fidelity assay for thermostable polymerases
• Secondary structure analysis of RNA
• Analysis of dsRNA molecules
• Thermal stability analysis of proteins
• Protein/protein or protein/ligand interaction analysis
1.3 Special features
1.3.1
Peltier powered linear temperature gradient
The heart of the TGGE MAXI system is the temperature block which is powered by peltier
technology. Thanks to precise microprocessor control a linear temperature gradient is
generated providing maximum reproducibility. Thus assay conditions can be much better
controlled compared to conventional chemical gradients (DGGE) or temporal gradients using
water baths.
The Biometra TGGE system is available in two formats. The standard TGGE “mini” system
operates small gels and is therefore ideally suited for fast, serial experiments. The TGGE
maxi system provides a large separation distance and allows high parallel sample
throughput.
Using the Biometra TGGE system it is very easy to separate samples either parallel or
perpendicular to a temperature gradient. All that has to be changed is the position of the
buffer tanks. Whereas perpendicular TGGE is mainly used for the optimization of separation
conditions, parallel TGGE allows fast analysis of multiple samples.
Perpendicular TGGE
Temperature gradient is perpendicular to the electrophoretic
migration:
One sample is separated over a broad temperature range to
determine the optimum temperature gradient or to analyze
temperature dependent changes in conformation
Parallel TGGE
Temperature gradient is parallel to electrophoretic migration:
multiple samples are separated in parallel
Instruction Manual TGGE MAXI 05/2012
7
Figure 2: Typical results after perpendicular TGGE (A: temperature gradient from left to right) and parallel TGGE
(B: temperature gradient from top to bottom).
1.3.2
Sensitivity
Because of the small amount of material used for separation, DNA or RNA fragments appear
as fine bands which can be clearly distinguished from each other. Even complex band
patterns can be analyzed due to the high resolution capability of the gradient block.
Comparing the TGGE method with other screening methods like SSCP the superior
performance of the TGGE method becomes evident.
In contrast to direct sequencing TGGE also detects mutations in mixed DNA samples.
Whenever heterozygous DNA is to be analyzed, direct sequencing will not give a clear signal
at the position of the mutation. This is especially the case if the mutated gene is masked by a
high background of normal cells. TGGE reliably detects mutations in a 1:10 dilution (and
higher) of wildtype DNA.
1.3.3
Patented technology
TGGE is protected by patents in most countries of the world. The patent for the TGGE
method is held by Qiagen AG, Hilden. Biometra is the exclusive licensee for manufacturing
and distribution of TGGE instrumentation.
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Instruction Manual TGGE MAXI 05/2012
1.4 Technical specifications
TGGE MAXI System
Electrophoresis unit with temperature gradient block and two
removable buffer chambers, controller, power supply, starter kit.
TGGE MAXI Electrophoresis
unit
Temperature gradient formation
High performance Peltier technology
Block size
20 x 20 cm
Temperature range
5 – 80 °C
Linear temperature gradient
maximum 45 °C
Temperature accuracy
+ 0.3 °C
Temperature uniformity
+ 0.5 °C
Glass plate size
23.5 x 23.5 cm
Gel size
approx. 20 x 20 cm
Separation distance
parallel: 16 cm
perpendicular: 19 cm
Dimensions (L x W x H)
42.3 x 42.3 x 33.3 cm
Weight
22 kg
TGGE MAXI System Controller
Microprocessor driven control of temperature gradient and
electrophoretic parameters
Current
maximum 500 mA
Voltage
maximum 400V
Wattage
maximum 50W
Program stores
up to 100 programs can be stored
Program modes
constant Voltage
V/h integration
Programs can contain different steps (pre-run, pause, run)
Display
LCD display, 4 lines, English / German
Interfaces
parallel (Centronics), serial (RS 232)
Dimensions
31 x 22 x 11.5 cm
Weight
3.5 kg
Voltage
110 / 230 V
TGGE MAXI Power Supply
Dimensions
29.5 x 22 x 8 cm
Weight
6.5 kg
Transformer
450 VA
Instruction Manual TGGE MAXI 05/2012
9
1.5 Legal Notes
1.5.1
Copyright
All rights reserved. It is not allowed to copy and publish the manual or parts of it in any form
as copies, micro film or other methods without a written authorisation from Biometra.
Biometra is pointing out that applied company and brand names are usually protected trade
marks.
The TGGE method is covered by patents issued to Diagen (now QIAGEN GmbH).
The polymerase chain reaction (PCR) process is covered by patents issued to Hoffman-La
Roche.
Acryl-GlideTM is a trademark of Amresco Inc.
Biometra is a trademark of Biometra GmbH.
The POLAND software service established by Gerhard Steger, Department of Biophysics,
University of Duesseldorf, is available by internet:
www.biophys.uni-duesseldorf.de/POLAND/poland.html
1.5.2
Liability
Biometra is not liable for damages and injuries caused by use not considering these
operating instructions in parts or completely.
1.5.3
Meaning of the Instructions
Biometra recommends that you first read these instructions carefully. This operation
instruction is part of the product and should be kept over the full life-time of the instrument. It
should also be forwarded to subsequent owners and users. Make sure that additions and
updates are inserted into the operation instructions.
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Instruction Manual TGGE MAXI 05/2012
2.
Safety and Warning Notices
2.1 Definition of Symbols
Symbol
Definition
Caution! Refer to instruction manual!
Danger! High voltage!
Fragile!
Danger! Hot surface!
2.2 Safety instructions / general remarks
The thermoblock will reach high temperatures during operation. The
thermoblock can burn you.
The TGGE system contains no user serviceable parts. Do not open
the housing instrument. Service and repair may only be carried out
by the Biometra Service department or otherwise qualified technical
personal.
Do not use the instrument when damages of the housing, block,
cable or other parts are visible.
Prior to connecting the unit to the power source please ensure that
the voltage setting at the fuse holder at backside of the power supply
is set to the required voltage.
Unplug the power cable before you open the TGGE system. Danger
of electric shock!
The thermoblock is covered with Teflon film. Avoid damaging this
film.
Make sure that the appliance connector and the plug of the supply
cord are accessible, so you can separate the instrument from the
mains.
Connect the TGGE system to a grounded socket.
Appropriate safety regulations must be observed when working with
infectious, pathogenic or radioactive material. Ask the responsible
local safety inspector for details.
Place the TGGE system on a stable, non flammable surface in a dry,
safe environment.
Instruction Manual TGGE MAXI 05/2012
11
Ensure that both the ventilation slits of the TGGE block not clogged
by dust or other material. Danger of overheating!
Do not fill buffer chambers above marking for maximum level
Do not move instrument during operation
In case of strong condensation under the safety lid stop run, dry
instrument and re-start
If buffer has been spilled on the electrophoresis unit, clean it
carefully before start of electrophoresis
Do not use paraffin oil on the thermoblock.
Switch off power before removing the safety lid
This instrument is designed and certified to meet EN 61010-1 safety
standards. It should not be modified or altered in any way. Alteration
of this instrument will void the warranty, void the EN61010-1
certification, and create a potential safety hazard.
Do not use alcohol (e.g. methanol, ethanol), organic solvents or
abrasives to clean the instrument.
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Instruction Manual TGGE MAXI 05/2012
3. Installation
3.1 Content of delivery
1)
Electrophoresis unit
including thermoblock, buffer chambers, safety lid
2)
Controller
control of electrophoresis parameters (Voltage) and temperature gradient
3)
Power supply
power supply for electrophoresis unit and controller
4)
Starter Kit
Glass plates, sealings, cover films, polybond films, buffer wicks, binder clips,
applicator strip, sample Acryl GlideTM* (*only in Germany)
5)
Manual
Please keep the original packaging material for return shipment in case of servicing.
TGGE MAXI System, 230/115 V (Germany only)
Electrophoresis unit with high precision gradient block, 2 buffer chambers for
variable positioning, controller, power supply, manual, TGGE MAXI Starter kit
024-204
024-200
TGGE MAXI System, 230/115 V (International)
Electrophoresis unit with high precision gradient block, 2 buffer chambers for
variable positioning, controller, power supply, manual, TGGE MAXI Starter kit
024-294
024-290
3.2 Unpack and Check
Unpack and carefully examine the instrument. Report any damage to Biometra. Do not
attempt to operate this device if physical damage is present.
Please keep the original packing material for return shipment in case of service issues
!! Attention !!
Please fill out and send back the warranty registration card. This is important for you
to claim full warranty.
3.3 Installation Conditions
•
Place the TGGE system on a stable surface in a dry, safe environment.
•
Let equilibrate the TGGE system to room temperature before starting operation.
•
Make sure that the appliance connector and the plug of the supply cord are accessible,
so you can separate the instrument from the mains.
•
Connect the TGGE system to a grounded socket.
Instruction Manual TGGE MAXI 05/2012
13
Ensure that the ventilation slits at the electrophoresis unit
are unobstructed.
Insufficient ventilation can cause overheating of the
instrument.
Prior to connecting the unit to the power source please ensure that the
voltage selector at the back side of the power supply is set to the
required voltage.
Danger of electric shock! Unplug the power cable before you open the
TGGE system.
3.4 Operation Voltage
Important: Prior to connecting the TGGE system to the mains, make sure that the voltage
setting of the power supply is in accordance with your mains voltage. The set operating
voltage is displayed in the small window of the fuse holder at the backside of the power
supply.
The TGGE system can operate at 110 or 230 Volt. To change operation voltage of the TGGE
system, switch off the instrument and disconnect the mains plug. Press the two clips both
ends of the fuse holder and excerpt it. Pull out the cream coloured insert from the fuse holder
and turn it by 180°. Insert it again and control the voltage displayed in the small window of
the fuse holder. If the setting is correct insert the fuse holder to the power supply again.
Backside power supply
Mains
inlet
Netzstecker
Sicherungen
Fuse
s
Fuse holder Netzschalter
Figure 3. TGGE system fuse holder
3.5 Setup
1) Connect electrophoresis unit and controller.
2) Connect controller and power supply
3) Connect power cables from the safety lid to the controller
The buffer chambers can be placed in two orientations, depending on the direction of the
temperature gradient. Be sure that the orientation is correct. The markings on the gradient
block (L0 to L10) indicate the direction of the temperature gradient. The direction of
electrophoresis (minus to plus for nucleic acids) is indicated on the safety lid.
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Instruction Manual TGGE MAXI 05/2012
4. Considerations for successful TGGE experiments
TGGE is a powerful technique to separate molecules of same size, but different sequence.
Nevertheless, every DNA fragment has its own characteristics and three steps have to be
taken before successful analysis of multiple samples in parallel TGGE can begin. Each of the
following steps is described in detail in section.
Step 1 Check the sequence of your PCR fragment in Poland analysis. The Poland
computer program (http://www.biophys.uni-duesseldorf.de/POLAND/poland.html) calculates
the melting behavior of dsDNA molecules. Poland analysis can predict, whether a fragment
is suited for TGGE or not. Analysis is available online and free of charge (see section 4.3.1).
Melting profile is ok
Poland analysis shows
a satisfying profile.
Proceed with step 2
Melting profile is not ok
If Poland analysis shows that the fragment in its current
state is not suited for TGGE, optimize your primer design.
Never try to separate samples in TGGE if the calculated
melting profile is not ok. Back to step 1
Step 2 If the Poland analysis shows a suitable melting profile you should test separation
conditions in a perpendicular TGGE. In perpendicular TGGE, a large aliquot of the sample
runs over a broad temperature range. The result of parallel TGGE allows identification of the
temperature gradient for parallel analysis.
Perpendicular gel is
ok
Perpendicular TGGE
shows a nice melting
curve.
Proceed with step 3
Perpendicular gel is not ok
If perpendicular analysis does not show the expected
melting profile, check sequence again in Poland analysis.
Also check purity of chemicals and electrophoretic
conditions.
Do not try samples is parallel TGGE, as long as the
perpendicular gel does not show a defined melting curve.
Back to step 2
Step 3 Set up a parallel gel with the temperature gradient derived from the perpendicular
gel. Separation can be optimized by varying the temperature gradient and voltage.
4.1 Adaptation of protocols from the TGGE “mini” system
In contrast to the „mini“ system, the TGGE maxi system works with gels of 1mm thickness.
Therefore electrophoretic parameters have to be adapted. This can be done by running a
perpendicular gel or by performing a time chase experiment. Both techniques are described
in detail in section 4.4 and 4.5.
Instruction Manual TGGE MAXI 05/2012
15
With the TGGE maxi system the settings for T1 and T2 have been omitted. The temperature
gradient is set by defining a temperature for L0 and a temperature for L10. The
corresponding lines are marked on the thermoblock. This distance between L0 and L10 is
effectively used for the separation. For the evaluation of stained gels we have included a
plastic film where lines L0 to L10 are indicated. The gel is placed on the film and the position
of the bands can be identified in correlation to the temperature lines (see section 6.2.7).
4.2 Design of TGGE experiments
There are 3 steps in the setup of a new TGGE experiment:
1) Design of the PCR fragment
2) Identification of the correct temperature gradient
3) Parallel analysis of multiple samples
4.3 Design of DNA fragment for TGGE
The design of the DNA fragment is an important step for successful TGGE. Starting with the
gene fragment of interest PCR primers should be designed with a conventional computer
program. The melting behaviour of the resulting fragment should then be checked with the
Poland software. It is essential that the DNA fragment shows different melting domains. If
there is only one single melting domain, an artificial higher melting domain (called GC clamp)
must be added during PCR.
4.3.1
Poland analysis
The melting profile of a DNA fragment can be analyzed with a computer program. The
Poland software calculates the melting behaviour of a DNA fragment according to its base
sequence. This software is free accessible via the internet.
(http://www.biophys.uni-duesseldorf.de/POLAND/poland.html).
How to perform a Poland analysis
•
Open start page (URL see above)
•
1) enter a name for the query
•
2) copy / paste DNA sequence in the sequence window
•
3) choose the Tm plot (de-activate all other plots)
•
4) submit query
•
5) retrieve Tm plot (melting curve)
Poland service request form
The Poland server will calculate the thermal denaturation profile of double-stranded RNA,
DNA or RNA/DNA-hybrids based on sequence input and parameter settings in this form.
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Instruction Manual TGGE MAXI 05/2012
NEW: Thermodynamic parameters set for dsDNA in 75 mM NaCl (Blake & Delcourt) added.
Calculation is based on Poland's algorithm in the implementation described by Steger.
Graphics results are directly sent to your WWW client.
1)
2)
Sequence title line:
Sequence:
(plain format;
no numbers;
max. 1000 nts;
min. 5 nts)
Mismatched positions:
(comma-separated numbers)
Thermodynamic parameters:
Dissociation constant ß:
Oligonucleotide
(ß is function of seq.length)
Long double strand
(default: ß=1.0E-3/M)
Strand concentration:
(default: 1.0E-6 M)
Temperature range:
Instruction Manual TGGE MAXI 05/2012
High temperature
Temperature
Low temperature limit:
limit: (default: 110.0 step size:
(default: 40.0 °C)
°C)
(default: 2.0 °C)
17
Tm(p=50%) plot 3d plot Mobility plot
3)
Which graphics do you want:
Melting
curve
Diff. melting
curve
Graphics size:
(GIF format)
4)
Click here to
Click here to
submit
, or
Zurücksetzen
the form to defaults.
The Tm plot (second order, red color) shows the melting profile of the DNA fragment
according to the base sequence. The ideal fragment shows at least two distinct melting
domains. Note that mutations can be detected in all but the highest melting domain. This
means that in a DNA fragment with two melting domains, mutations can only be detected in
the lower melting domain.
Figure 4: Tm plot of a 140bp DNA fragment resulting from Poland analysis. The second order curve (red color in
the original) shows two different melting domains.
If the fragment consists of a single melting domain only, or if you want to scan the entire
fragment for mutations, add a so called GC clamp to one end of the PCR fragment.
4.3.2
GC clamps
A GC clamp is an artificial, high melting domain which is attached to one end of the fragment
during PCR. The name “GC clamp” implies that this short stretch will hold the DNA fragment
together, preventing a dissociation into the single strands at higher temperatures. The
optimum location for the GC clamp at the PCR fragment (5´ or 3´) can be easily checked with
the Poland software. Copy / paste the GC sequence to either side of your sequence and
repeat Poland analysis. In the following box you will find different examples for a GC clamp.
short GC-clamp (23 bp):
18
cccgc cgcgc cccgc cgccc gcc
Instruction Manual TGGE MAXI 05/2012
long GC-clamp (40 bp)44:
cgccc gccgc gcccc gcgcc cggcc cgccg ccccc gcccg
long GC-camp (39 bp)45:
ccccg ccccc gccgc ccccc ccgcg cccgg cgccc ccgc
To integrate a GC clamp into a PCR fragment, one of the two primers has to be modified.
The non-specific GC sequence is added to the 5´-end of the primer. Thus the GC sequence
is incorporated in the fragment during PCR. Prior to order primers check the melting
behaviour of the PCR fragment with GC clamp by Poland analysis (see chapter 4.3.1).
Figure 5 shows a Tm plot for a PCR fragment with GC clamp either at the 5’-or 3’-end.
GC-clamp at 5´end
GC-clamp at 3´end
bubble
Figure 5: Tm-Plot of a PCR fragment with GC clamp at the 5’-end or at the 3’-end. Whereas the fragment with 5’GC clamp has domains that melt consecutively and is well suited for TGGE analysis the fragment with 3’-GC
clamp has a domain in the middle that melts at lower temperature compared to the neighbouring domains and is
therefore leading to bubble formation. Thus the 3’-GC clamp for this fragment will not work for TGGE analysis.
4.3.3
Chemical clamp with Psoralen (Furo[[3,2-g]]coumarin, C11H6O3)
In addition to “clamping” a fragment with an artificial high melting domain it is as well possible
to covalently fix the end of a PCR fragment. To achieve this, one of the primers carries a
Psoralen molecule. Psoralen is a high reactive group when exposed to UV radiation. Thus it
is possible to covalently close one end of the PCR fragment. The optimal primer sequence
may be 5’(Pso)pTaPpnpnp.....3’, given the preference of Psoralen for binding between TpA
and ApT pairs 13,46,47. Crosslinking of the PCR product is done e.g. in a flat-bottom
microtiter plate using a 365 nm UV source. Working with small volumes it may be necessary
to minimize evaporation by cross-linking at 4 – 10°C. The yield is not affected by
temperature. The distance of the sample from the UV source affects the yield. 15 min at 0.5
cm distance of the sample from an 8 W UV lamp is sufficient.
4.3.4
Use of SSCP primers
In many cases primer from SSCP may be used for TGGE analysis. Nevertheless, we highly
recommend to check SSCP fragments including primer sequences via Poland analysis first
since the fragment might not be suited for TGGE analysis. For example, a mutation occurring
in the high melting domain might lead to non-interpretable results. If there is only one melting
domain add a GC clamp to one of the primers (see section 4.3.2)
Instruction Manual TGGE MAXI 05/2012
19
4.4 Find correct temperature gradient
Poland analysis gives the first indication, which temperature gradient should be applied for
parallel analysis of multiple samples. Under experimental conditions separation is performed
in the presence of high concentrations of urea. Urea lowers the melting temperature of the
DNA. This is important because gel electrophoresis at very high temperatures may lead to
partial drying of the gel, resulting in a disturbed separation pattern. Therefore it is necessary
to identify the optimum temperature gradient under experimental conditions.
To identify the optimum temperature gradient the DNA fragment is separated in a
perpendicular TGGE. This means the temperature gradient is perpendicular to the migration
of samples (see section 6.2.5). Thus the migration of a fragment can be checked
simultaneously at different temperatures in a single run. If the PCR fragment has been
designed properly the separation in a perpendicular temperature gradient leads to a distinct
melting curve (see Figure 6)
Figure 6: Identification of the optimum temperature gradient in a perpendicular TGGE. At low temperature (below
T1) DNA migrates as a double strand (left side). At intermediate temperature (between T1 and T2) the DNA
opens at one side, the partial double strand is increasingly slowed down. Above T2 the DNA separates into the
single strands.
At T1 the double strand starts to melt and forms a branched structure. At T2 the partial
double strand separates irreversibly into the single strands. Analysis of samples in parallel
TGGE should be performed precisely in this temperature range between T1 and T2.
How to identify the optimum temperature range from a perpendicular gel:
Place the stained gel on the plastic film with the printed lines (l0 to L10). Identify the line
where the double strand starts to melt (T1) and the line where the double strands separates
into the single strands (T2). For the calculation of temperature at the corresponding lines see
section 6.2.7.
4.5 Parallel analysis of multiple samples
After identification of T1 and T2 in a perpendicular TGGE this temperature gradient is spread
over the whole block for parallel analysis.
20
Instruction Manual TGGE MAXI 05/2012
electrophoresis
cold
warm
T1
T2
Figure 7: Application of T1 and T2 in a parallel gel.
Note: The DNA fragments are separated by their melting behavior. They can be
distinguished as soon as the fragments begin to melt, i.e. they form a fork like structure
(temperature higher than T1). During electrophoresis the fragments should not separate into
single strands. This is an irreversible transition resulting in diffuse bands.
Note: If there are only small differences in the migration of different samples, perform a
heteroduplex analysis (see chapter 9.3)
Instruction Manual TGGE MAXI 05/2012
21
5. Sample preparation
5.1 Purity of samples
Due to the high sensitivity of the staining procedure after TGGE it is recommended to use
purified DNA, RNA or protein samples. Any impurities might be misinterpreted after TGGE,
thereby making the analysis of gels difficult. Nevertheless it is possible to use even crude
mixtures for TGGE analysis.
PCR-amplified DNA fragments usually can be analyzed without further purification. Please
note that the presence of high amounts of nonspecific, secondary PCR products may result
in difficulties with interpretation of band pattern, melting profile, etc. For example, in parallel
TGGE, nonspecific bands with a higher molecular weight than the specific PCR product may
be misinterpreted as heteroduplices, or analogs with lower thermal stabilities. Therefore,
prior to TGGE check the PCR product in a conventional agarose gel. If necessary, purify
your specific PCR product, e.g., by agarose gel electrophoresis and subsequent gel
extraction.
5.2 Sample preparation for direct DNA analysis
1 volume of DNA/RNA samples is mixed with 1 volume of TBE or Na-TAE loading buffer or
with 0.1 volume of the total loading volume ME loading buffer (see Appendix). The resulting
mixture is loaded directly on to the polyacrylamid gels. Be sure that the slots are filled up to
maximum (if necessary, add 1x loading buffer to fill up the slots to maximum).
In case of low-concentration samples we recommend to prepare 5x conc. loading buffer. 0.2
volume of this concentrated loading buffer is mixed with 0.8 volumes of the sample and
loaded onto the gel.
5.3 Denaturation / Renaturation for heteroduplex analysis of DNA
Mix sample with equal amount of standard DNA and heat to 95°C for 5 minutes
(denaturation). Then let slowly cool down to 50°C (renaturation). This can be done by
programming a thermocycler to 94°C for 5 minutes and then 50 °C for 15 minutes with a
ramping rate of –0.1°C/second. The sample is then loaded directly to the gel. In order to
achieve the recommended loading volumes for diagonal or perpendicular TGGE , the sample
volume should be adjusted with running buffer.
Important: Do not denature/renature DNA for community profiling (see chapter 6.1.3)!
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Instruction Manual TGGE MAXI 05/2012
6. Operating
6.1 Casting of gels
6.1.1
Assembly of the gel cassette
There are two different types of gels for TGGE. For perpendicular gels one long slot is used
to separate one sample over a broad temperature range (use glass plate with slot former,
024-228). For parallel gels samples are applied with an applicator strip. For use with this strip
a gel without slots is casted (use glass plate without slot formers, 024-227), then the
applicator strip is applied onto the gel just prior to run. This silicone applicator strip contains
32 holes and is simply placed on top of the gel. Samples are loaded in the holes. After
turning on voltage samples diffuse into the gel and migrate along the gel matrix.
Slotformer
Glass plate
for parallel gel
024-227
(no slotformer)
Spacer
Glass plate
for perpendicular gel
024-228
(with slotformer)
Figure 8: Glass plates for casting parallel (left) or perpendicular gels (right).
The gel cassette consists of one glass plate with spacers (024-227 or 024-228), glass plate
without spacers (024-221) and a silicone sealing (024-230). The sandwich is fixed with 9
metal clamps.
Alternatively the glass plate with spacers and slots (024-229, not supplied) can be used to
assembly the gel cuvette.
Instruction Manual TGGE MAXI 05/2012
23
Glass plate without
spacers
Polybond Film
Glass plate with
spacers
and silicone sealing
Figure 9: Setup of glass plate sandwich
Note: The gel is poured on Polybond film to optimize temperature transition between block
and gel. The gel is bound covalently to this hydrophilic Polybond film (024-234).
Note: The gel sticks to the Polybond film throughout the whole procedure, including staining.
1)
Clean both glass plates with 70% ethanol and a soft tissue.
2)
Apply approx. 2 ml of gel repellent solution (Acryl Glide, Amresco or Repel silane,
Sigma) on the glass plate WITH SPACERS and spread it with a soft tissue
3)
Wait for 2 minutes
Polish glass plate with a soft tissue
Note: Do not apply Acryl-Glide onto the spacers, because this can lead to leakage of the gel
cuvette.
4)
Put polybond film (024-234) on the glass plate WITHOUT SPACERS.
5)
Attach polybond film by carefully wiping it with a soft tissue. The film should attach
uniformly to the glass plate. To improve contact between glass plate and film a drop
of water may be applied behind the Polybond film.
Note: The support film may be fixed along the upper side of the cover glass plate with
ordinary adhesive tape. This way no gel solution can accidentally get behind the support film.
6)
24
Place silicone sealing around spacers.
Instruction Manual TGGE MAXI 05/2012
Glass plate with
spacers
Silicone sealing
Figure 10: Assembly of glass plate and sealing
7)
Assemble gel sandwich and fix it with 3 metal clamps on each side.
Figure 11: Final setup of the gel cuvette.
Note: the clamps should be placed directly on the spacers.
8)
Set gel sandwich upright on the 3 bottom clamps (see Figure 11)
6.1.2
Preparing gel solution for mutation analysis
The choice of the buffer system has a strong impact on TGGE analysis. Concentration of salt
and denaturing agents (urea or formamide) strongly affects the melting temperature of DNA
and proteins. In general, urea is used for the separation of nucleic acids in a concentration
between 7 and 8M. Urea reduces the melting temperature and thus enables a separation at
lower temperatures (which is favourable, because at higher temperatures the gel tends to dry
out). To further reduce the melting temperature (deionized) formamide may be used in
concentrations of up to 20%. The most popular buffer systems for TGGE are TBE, TAE and
MOPS (see also chapter 17.2). In the following paragraph a standard protocol for the TAE
buffer system is listed. Please note, that the buffer system should be adapted to each special
kind of application. In chapter 6.1.3 the gel composition for the separation of fragments from
mixed populations is given.
Instruction Manual TGGE MAXI 05/2012
25
For one TGGE MAXI gel prepare 50 ml gel solution
TAE gel composition
final concentrations
stock solution
for 50 ml
for 100 ml
Acrylamide [8%]
40 % (37,5 :1)
10 ml
20ml
Urea [8M]
solid
24 g
48 g
TAE [1x]
10 x
5 ml
10 ml
Glycerol [2%]
40%
2,5 ml
5 ml
to 50 ml
to 100ml
Adjust with aqua bidest
•
Stir solution at 50°C until urea is completely dissolved.
•
Carefully degas gel solution
•
let cool down to room temperature and start polymerization with
APS
10 %
80 µl
160 µl
TEMED
100%
45 µl
90 µl
•
Load gel solution in a syringe and attach a 0.4µm or 0.25µm sterile filter
•
Pour gel through sterile filter into the glass sandwich.
Protocols for mutation analysis are given in chapter 7.3.
6.1.3
Gel preparation for analysis of mixed bacterial populations (genetic fingerprint)
For genetic fingerprinting primers against 16SrRNA genes are used. Depending on the
complexity of the sample and the primer design quite a high number of PCR fragments can
arise from one sample. In this regard there are two major differences to mutation analysis: 1)
the number of fragments per lane is much higher 2) the amount of DNA per band is lower. To
separate all the many fragments a rather flat gradient is used which has to be optimized very
carefully. For detecting low amounts of DNA a clear staining background is essential. The
silver staining protocol (chapter 8.1.) has been optimized for gels with high content of urea
and formamide and thus achieves high sensitivity at low background.
For microbial diversity analysis we recommend the following conditions. However, these
parameters can only be the starting point for individual optimization.
TAE gel composition
final concentrations
stock solution
for 50 ml
for 100 ml
Acrylamide [6%]
40 % (37,5 :1)
7,5 ml
15 ml
Urea [8M]
solid
24 g
48 g
TAE [1x]
10 x
5 ml
10 ml
Glycerol [2%]
40%
2,5 ml
5 ml
Deionized formamide [100%]
20%
10 ml
20 ml
to 50 ml
to 100ml
Adjust with aqua bidest
•
26
Stir solution at 50°C until urea is completely dissolved.
Instruction Manual TGGE MAXI 05/2012
•
Carefully degas gel solution
•
let cool down to room temperature and start polymerization with
APS
10 %
80 µl
160 µl
TEMED
100%
45 µl
90 µl
•
Load gel solution in a syringe and attach a 0.4µm or 0.25µm sterile filter
•
Pour gel through sterile filter into the glass sandwich.
Protocols for mutation analysis are given in chapter 7.4.
Important: Do not denature/renature the DNA fragments prior to electrophoresis!
Tip: Formamide can be a "tricky" component since it doesn't have the highest stability in
general. You might even leave formamide in the experiments - a test with and without
formamide will show whether the fragments also become separated without this component
at the same resolution. The preparation 20 ml aliquots that are stored at -20 °C is
recommendable.
6.1.4
Pouring gels
1) Pour gel solution slowly into the sandwich. Avoid bubbles!
2) Let polymerize for approx. 3 h at room temperature
Note: Polymerized gels may be stored for 3 days or even longer at room temperature.
Remove clamps and wrap gel sandwich including glass plates in wet paper towels. Store the
sandwich in a tight plastic bag.
Note: Prepare electrophoresis unit prior to disassembling the gel cassette. The gel should
not be exposed to the air for extended periods since this may lead to drying of the gel.
3) After polymerization remove clamps. Remove the glass plate without spacers by sliding
the glass plate away from the rest of the sandwich (if you have fixed the support film with
adhesive tape, remove or cut tape first). The gel must stick on the support film.
4) Remove gel together with support film carefully from the glass plate with spacers. Be
careful not to damage the slots.
6.2 Electrophoresis
6.2.1
Electrophoresis conditions
The electrophoresis unit of the TGGE System has been designed to accommodate TGGE
and all related applications like CTGE, TTGE and SSCP without cumbersome changes. The
buffer tanks can be positioned in two orientations, allowing a temperature gradient parallel or
perpendicular to the electrophoresis direction (see section 6.2.5 and 6.2.6).
Electrophoresis conditions in general depend on
•
the kind of sample, e.g. protein, nucleic acid, fragment size
•
the kind of application, e.g. parallel or perpendicular TGGE
•
the sample preparation, e.g. high salt or low salt preparation,
Instruction Manual TGGE MAXI 05/2012
27
•
the buffer system.
Any recommendations should be regarded as guidelines to start with. Further improvement
of the analysis should be done by adjusting the run conditions to individual needs.
Important: The controller is designed to control voltage rather than amperage (set [mA] and
[Vh] to maximum values).
m can be set up in the controller
6.2.2
Setup electrophoresis unit
The Biometra TGGE system is a horizontal electrophoresis system. The buffer bridges to the
gel are established by layering one side of each buffer wick (024-215) on the gel and
submerging the other in the buffer inside the tank. To protect the gel from drying, it is
covered with a gel cover film (024-232). The complete setup, consisting of gel with cover film
and buffer wicks, is covered with the gel cover plate. The gel cover plate has two sealings
and fits tightly onto the thermoblock. It holds the buffer wicks in place and helps to build a
humidity chamber around the gel. This is important to prevent evaporation during the run.
Important: Never run a gel without gel cover plate. (This could lead to
massive condensation under the safety lid. Danger of electric shock.)
Gel cover plate
Sample
Figure 12: Set up of the gel for electrophoresis
6.2.3
Prior to assembly of the electrophoresis unit
Note: Be sure to have everything on hand, to avoid extended handling times. Don’t let the
disassembled gel dry, during setup of the electrophoresis system.
Prepare:
parallel or perpendicular gel (see section 4.2)
samples in loading buffer (for sample preparation see chapter 5)
28
Instruction Manual TGGE MAXI 05/2012
1.000 ml running buffer (for recipes see chapter 17.2)
2 re-usable buffer wicks (024-216)
1 cover film (024-232)
thermal coupling solution (0.1% Triton X-100 or 0.1% Tween 20, degas carefully)
gel cover plate
6.2.4
Gel setup for electrophoresis
1)
Adjust electrophoresis chamber with the 4 levelling feet.
2)
Fill 500 ml running buffer in each buffer tank (check orientation of the buffer tanks:
parallel or perpendicular TGGE! see section 6.2.5 and 6.2.6).
Note: Wipe off any spilled buffer from the electrophoresis unit. Never run device if buffer
has been spilled.
3)
Soak 2 buffer wicks with running buffer
4)
Disassemble gel sandwich. Carefully clean backside of the gel support film with a
soft tissue.
5)
Apply not more than 1.5 ml of thermal coupling solution (0.1% Triton X-100 or 0.1%
Tween 20) on the thermoblock
Note: The volume of coupling solution should be as small as possible. Excess coupling
solution leads to an irreproducible temperature distribution under the gel. The result is a
wavelike migration front and poor separation of fragments.
6)
Place the gel on the thermoblock. The thermal coupling solution should spread over
the whole block. Avoid formation of bubbles. Wipe off any residual coupling solution
along the edges of the gel support film.
Note: The thermal coupling solution is essential for efficient heat transfer from block to gel. If
bubbles are entrapped under the gel support film, remove support film with gel from the block
and place it back again on the block.
7)
Apply applicator strip for sample loading
8)
Cover the gel just beneath applicator strip with a cover film.
9)
Attach pre-soaked buffer wicks on top and bottom of the gel.
Instruction Manual TGGE MAXI 05/2012
29
Buffer wick
Gel
Cover film
Gel
Buffer wick
Figure 13: Setup of gel, cover film and buffer wicks.
10)
Load samples (approx. 5µl each for parallel gel with 32 slots, approx. 200µl for
perpendicular gel with)
Note: Be careful not to touch the samples with the buffer wick! Otherwise the samples will
diffuse into the wick.
11)
Attach gel cover plate (the cover plate should have contact to the wicks, but must not
squeeze gel or wicks).
12)
Close safety lid and start run.
Note: For parallel TGGE let temperature gradient equilibrate for approx. 10 minutes, then
start main run. This step may be omitted for a perpendicular gel.
6.2.5
Perpendicular TGGE
In perpendicular TGGE one sample is separated over a broad temperature range. This
application is mainly used to check the melting behaviour of a sample (see section 4.4). For
casting of the gel use a glass plate with one large slot former (024-228). The temperature
gradient must be orientated perpendicular to the migration of the sample. The buffer tanks
must be positioned as described in Figure 14.
The migration of DNA / RNA molecules is indicated by the arrow on the safety lid of the
electrophoresis unit.
30
Instruction Manual TGGE MAXI 05/2012
Figure 14: Positioning of buffer tanks for perpendicular TGGE. Be sure that the direction of electrophoresis is
perpendicular to the temperature gradient. The temperature gradient is indicated by the lines on the edge of the
block.
6.2.6
Parallel TGGE
In parallel TGGE multiple samples are separated along the temperature gradient. For gel
casting gel use glass plate with spacers (024-227). The buffer tanks for parallel TGGE must
be positioned as depicted in Figure 15.
Figure 15: Positioning of buffer tanks for parallel TGGE. Be sure that the direction of electrophoresis is parallel to
the temperature gradient. The slots of the gel should be at the same side as the markings on the block.
Note: For a parallel TGGE a 10 minutes equilibration of the temperature gradient may be
included after the pre-run.
6.2.7
How to identify the optimum temperature range from a perpendicular gel
The theoretical background for the separation of DNA fragments in a perpendicular gel is
described in section 4.4.
Instruction Manual TGGE MAXI 05/2012
31
Place the stained perpendicular gel on the plastic film with the printed lines (L0 to L10).
Identify the line where the double strand starts to melt (T1) and the line where the double
strands separates into the single strands (T2).
0
1
2
3
4
5
6
7
8
9
1 0
Figure 16: The calculation of the corresponding temperatures is simple, since there is a linear temperature
gradient between L0 and L10 (i.e. the temperature increment from one line to next line is always the same).
Calculation: Divide range of gradient by ten, this is the temperature increment from one line
to the adjacent lane.
Example:
calculation of temperature at line 6 (L6) in a temperature gradient from 40°C
(L0) to 60°C (L10)
•
subtract temperature at L0 from temperature L10 (range of gradient: 60-40°C =
20°C)
•
divide temperature by 10 (increment from line to lane: 20°C/10 = 2°C)
•
multiply increment by 6 (6 increments from L0 to L6: 12°C)
•
add this value to the temperature at L0 (40°C + 12°C)
result:
32
temperature at L6 is 52°C
Instruction Manual TGGE MAXI 05/2012
7. Programming the TGGE controller
7.1 General overview
All parameters of the run are controlled by the TGGE system controller. This includes
electrophoretic parameters (voltage, amperage, time) as well as control of the temperature
gradient.
Note: Each program can consist of several steps. Thus you can program pre-run,
equilibration and main run in the same program (see also section).
7.1.1
Create / edit program
Main screen
L0: 20.0°C L10: 20.0°C
maxi block off
A? BElpho Cprograms D+
Press C [programs] to enter the programming mode
program no:
Alist Bdel Cquit Denter
7.1.2
Select program
Enter a program directly by number or press A [list] to view a list of the existing programs.
0 test 1
maxi
1 parallel mini
2 empty
A B C quit D enter
Scroll through the list with A
B
and accept highlighted program with D [enter].
Note: Each program depends on the type of TGGE system (mini or maxi). The type (mini or
maxi) for which the program was written, is displayed behind the program name. Programs
can only be written /edited in the respective mode. Programs that are created in the maxi
mode are automatically saved as maxi programs and can only be run with the maxi system
(and vice versa).
To set type of system (mini or maxi) see section 7.2.4.
Instruction Manual TGGE MAXI 05/2012
33
7.1.3
Name program
Each program is specified by name and a program number. To facilitate retrieval of a
program, you can enter a name for each program existing of letters, numbers and symbols.
name:>
<
ABCDEFGHIJKLMNOPQRST
UVWXYZ –()αδ/,〈〉&+.%
A BABC C quit D enter
Press B [ABC] to enter the mode for the selection of letters.
name:>
<
ABCDEFGHIJKLMNOPQRST
UVWXYZ –()αδ/,〈〉&+.%
A B C quit D enter
Move to the desired letter with A [] and B [
name:>test
]. Accept highlighted letter with D [enter].
<
ABCDEFGHIJKLMNOPQRST
UVWXYZ –()αδ/,〈〉&+.%
A B C quit D enter
If the program name is complete, confirm name with D [enter]. In the following screen you
can set the temperatures for the gradient block.
7.1.4
Enter temperatures for the gradient block
1:L0:
A? B
L10:
C quit D
Enter temperature for L0 and accept with D [enter].
1:L0:30.0
L10:
A? Bdelete Cquit Denter
Enter temperature for L10 and accept with D [enter].
1:L0: 30.0
L10: 70.0
ok?
A B no C quit D yes
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Instruction Manual TGGE MAXI 05/2012
Confirm settings with D [yes]. If settings are not correct press B [no] and repeat entry of
temperature settings.
After you have confirmed the temperature settings, the following screen is displayed. Here
you can enter all parameters for electrophoresis.
7.1.5
Enter electrophoresis parameters
1:L0: 30.0 L10: 70.0
time:
El: 0V 500mA 50W
A? B V*h C quit D
Enter time for electrophoresis and accept with D [enter].
Note: There is a convention on how time settings are entered in all BIOMETRA instruments:
hours
minutes
seconds
If you enter a number without “dot” this value will be interpreted as seconds (“300” => 5
minutes). To program minutes enter a “ ” after the number of minutes. To enter hours enter
after the number. You can also enter any combination of hours, minutes and seconds.
Example: for 1 hour, 30 minutes, 20 seconds enter 1 30 20.
The time values will be displayed in the following format: 00 m 00s
Accept time setting with [D enter]
1:L0: 30.0 L10: 70.0
time: 10m 0s
El: 0V 500mA 50W
A? Bdelete C quit D enter
Enter Voltage and accept with D [enter]
1:L0: 30.0 L10: 70.0
time: 10m 0s
El: 300V 500mA 50W
A? Bdelete C quit D enter
Note: The values for amperage [mA] and wattage [W] are set to maximum level as default. If
you enter lower values, these parameters may become limiting during electrophoresis.
Accept default settings for voltage and wattage with D [enter] or enter different values.
Note: We recommend to control electrophoresis by constant voltage rather than by constant
amperage (set [mA] to maximum value, respectively accept default value).
Note: Each program can consist of several steps. Thus it is possible to program complex
protocols including a pre-run, a pause for handling of the gel and the main run..
In the following screen you can program a second step for your protocol.
2:L0:
L10:
Instruction Manual TGGE MAXI 05/2012
35
A? B
C quit D
If you do not want to program another step, accept program with C [quit].
The program name, number of steps and the total run time is displayed.
program no. 8
pgm end: 1 step(s)
run time: 0h10m 0s
A? B
C quit D
7.1.6
Start electrophoresis
Main screen:
L0: 20.0°C L10: 20.0°C
maxi block off
A? BElpho Cprograms D+
To start a program press B [Elpho]
start program:
Alist Bdel Cquit Denter
Enter program number or choose a program from the list with A [list].
start program: 8
Alist Bdel Cquit Denter
Confirm program number with D [enter].
The program starts and parameters of gradient block and electrophoresis are displayed.
During temperature equilibration of the gradient block the elapsed time is displayed.
Note: Electrophoresis starts as soon as the set temperature in the block is achieved.
L0: 30.0°C L10: 70.0°C
hold: 1 2m12s 11.4Vh
El: 300V 8mA 20.3W
A? BElpho Cprograms D+
7.1.7
Stop/pause electrophoresis
L0: 30.0°C L10: 70.0°C
hold: 1 2m12s 11.4Vh
El: 300V 8mA 20.3W
A? BElpho Cprograms D+
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Instruction Manual TGGE MAXI 05/2012
To stop/pause the active program press B [Elpho]
program 8 test
pause ?
stop ?
A? Bpause Cquit Dstop
Press B [pause] to pause program
Press D [stop] to stop program
Press C [quit] to return to the active program.
7.1.8
View temperatures of the gradient
L0: 30.0°C L10: 70.0°C
hold: 1 2m12s 11.4Vh
El: 300V 8mA 20.3W
A? BElpho Cprograms D+
To display the temperatures in the block during a run press A [?]:
L0 : 30.1
L1 : 34.2
L2 : 38.3
A
B
C quit D enter
You can scroll through the different lines with A
B .
L6 : 54.8
L7 : 58.9
L8 : 62.9
A
B
C quit D enter
Note: There is a difference is the number of lines depending on the type of TGGE system
(mini or maxi) that is installed. In the maxi mode 10 different temperatures are displayed (L0
to L10) in the mini mode 5 different temperatures are displayed (L1 to L6).
7.2 Special functions
Main screen
L0: 20.0°C L10: 20.0°C
maxi block off
A? BElpho Cprograms D+
Press D [+] to enter the menu for special functions
1 print programs
2 signal
Instruction Manual TGGE MAXI 05/2012
37
3 language
A
B
C quit D enter
Scroll through the list with A
7.2.1
B .
Print programs
Connect controller to a dot matrix printer. Select option 1 in the above menu and confirm with
D [enter].
7.2.2
Select / de-select signal
Select option 2 in the special functions. Press A [on] to activate the signal, press B [off] to
inactivate signal.
7.2.3
Select language
Select option 3 in the special function screen. Choose between German and English
7.2.4
Set block type
Select option 6 in the special function screen. Choose between mini and maxi system.
Note: The selection of the TGGE system type (mini or maxi) is saved together with each
individual program. Programs that have been written in the MAXI mode can only be edited
and run in the maxi mode. Programs that have been written in the mini mode can only be
edited and run in the mini mode.
7.3 Electrophoresis protocols for mutation analysis
7.3.1
Electrophoresis protocol for perpendicular TGGE*
Step 1:
L0: 20.0 L10: 20.0
time: 10m 0s
El: 300V 500mA 50W
A? B V*h C quit D
Pre-run
Step 2:
L0: 30.0 L10: 70.0
time: 10m 0s
El: 0V 500mA 50W
A? B V*h C quit D
Equlibration phase of the gradient
Step 3:
38
Instruction Manual TGGE MAXI 05/2012
L0: 30.0 L10: 70.0
time: 3h 30m 0s
El: 300V 500mA 50W
A? B V*h C quit D
Main run
* all information provided has to be considered as starting point. Further adaptations might be
necessary
7.3.2
Electrophoresis protocol for parallel TGGE*
Step 1:
L0: 20.0 L10: 20.0
time: 10m 0s
El: 300V 500mA 50W
A? B V*h C quit D
Pre-run
Step 2:
L0: XX.X L10: YY.Y
time: 10m 0s
El: 0V 500mA 50W
A? B V*h C quit D
Equilibration phase of the gradient§
§§
XX.X = start of DNA melting* minus 5 °C
§§
YY.Y = end of DNA melting* plus 5 °C
§
determined in the perpendicular TGGE experiment
§§
safety margin (might not be required)
Step 3:
L0: XX.X L10: YY.Y
time: 4h 0m 0s
El: 300V 500mA 50W
A? B V*h C quit D
Main run
* all information provided has to be considered as starting point. Further adaptations might be
necessary
7.4 Electrophoresis protocols for mixed population analysis
7.4.1
Electrophoresis protocol for perpendicular TGGE*
Step 1:
L0: 20.0 L10: 20.0
time: 10m 0s
El: 300V 500mA 50W
A? B V*h C quit D
Pre-run
Step 2:
Instruction Manual TGGE MAXI 05/2012
39
L0: 30.0 L10: 70.0
time: 10m 0s
El: 0V 500mA 50W
A? B V*h C quit D
Equlibration phase of the gradient
Step 3:
L0: 30.0 L10: 70.0
time: 16h 0m 0s
El: 130V 500mA 50W
A? B V*h C quit D
Main run
* all information provided has to be considered as starting point. Further adaptations might be
necessary
7.4.2
Electrophoresis protocol for parallel TGGE*
Step 1:
L0: 20.0 L10: 20.0
time: 10m 0s
El: 300V 500mA 50W
A? B V*h C quit D
Pre-run
Step 2:
L0: XX.X L10: YY.Y
time: 10m 0s
El: 0V 500mA 50W
A? B V*h C quit D
Equilibration phase of the gradient§
§§
XX.X = start of DNA melting* minus 5 °C
§§
YY.Y = end of DNA melting* plus 5 °C
§
determined in the perpendicular TGGE experiment
§§
safety margin (might not be required)
Step 3:
L0: XX.X L10: YY.Y
time: 16h 0m 0s
El: 130V 500mA 50W
A? B V*h C quit D
* all information provided has to be considered as starting point. Further adaptations might be
necessary
40
Instruction Manual TGGE MAXI 05/2012
8. Staining of gels
8.1 Silver staining
Aside from autoradiography silver staining is the most sensitive method for detecting small
amounts of DNA, RNA or proteins in polyacrylamide gels. Other staining protocols may be
used, but generally exhibit less sensitivity. This must be considered in relation to the amount
of DNA loaded on the gel.
All incubation steps are done in small plastic containers which are agitated on a rocking
platform.
Wear non-powdered protective gloves during all steps of the silver staining protocol
to avoid staining artifacts due to the high sensitivity of the staining protocol.
The quality of chemicals is essential in silver staining. Prepare solutions fresh, use
only chemicals of high quality (p.a.) and fresh double distilled water.
Important: Remove the protective plastic sheets from the gel.
•
Carefully remove any residual thermal coupling solution from the back of the gel (gel
support film) prior to staining.
•
Put the polyacrylamid gel with the gel side upwards into the staining tray. Avoid air
bubbles during all staining steps.
•
It’s recommended to prepare at least 400 ml solution for each incubation step.
•
Prepare stopping solution prior to developing.
8.1.1
Silver staining protocol
Step
Time
Solutions
Fixation
30 min
300-400 ml
10% glacial acid, 30% EtOH
Sensitization
2 x 30 min
300-400ml
30% EtOH
Washing
Rins gel 30 seconds under
running water,
Silver Binding
then wash 5 x 10 min
Fresh aqua dest
30 min
400ml 0,1 % AgNO3, prepare freshly
add 400µl Formaldehyde (37%) prior to use
Washing
Rins 30 seconds
then wash 1 min
Rins again 30 seconds
Instruction Manual TGGE MAXI 05/2012
Fresh aqua dest.
41
Developing
Until bands become visible, Solution 1: dissolve 2g Sodium thiosulfate
(Na2S2O3) in 10ml bidest,
can take several minutes,
Solution 2: dissolve 10g Sodium Carbonate
don´t let gel unattended!
(Na2CO3) in 400ml bidest
Add 400µl solution 1 to solution 2
Add 400µl Formaldehyde (37%)
Stopping
30 min
Dissolve 5,84g EDTA and 8g Glycine in 400ml
bidest
Storage
Up to several days
10% Glycerol
8.2 Ethidium bromide-staining
Incubate the gel in staining solution (0.5 µg/ml ethidium bromide in 1 X TBE) for 30 - 45 min.
Analyze under UV radiation (27).
8.3 Autoradiography
TGGE gels can also be directly exposed to x-ray films if radiolabeled samples are analyzed.
Direct exposure:
Incubate the TGGE gel for 15 min. in Fixation solution (see 8.1 Silver staining). Optional:
Silver stain the gel.
Remove residual buffer from the gel. Expose to an x-ray film at room temperature.
Exposure of dried TGGE gels:
Incubate the TGGE gel for 15 min. in Fixation solution (see 8.1 Silver staining). Optional:
Silver stain the gel.
Incubate the gel in 2-5% glycerol for 10 minutes to prevent the gel from cracking. Incubate an
appropriate sheet of cellophane (no Saran wrap!!!!!) in 2 - 5% glycerol. Layer the cellophane
on the gel. Air dry the sandwich at room temperature for one day or use a gel dryer at 50°C
for at least 3h. Exposure to an X-ray film.
8.4 Elution of DNA from the TGGE gels
DNA fragments which have been separated on TGGE, for example, different alleles of one
gene, can be eluted from silver-stained TGGE gel and re-amplified by PCR.
Using a Pasteur pipette, puncture the gel and extract a µl piece containing the particular DNA
duplex. Incubate in 20 µl TE buffer overnight. Use a 1 µl aliquot for re-amplification.
42
Instruction Manual TGGE MAXI 05/2012
9. Trouble shooting guide
The following trouble-shooting guide may be helpful in solving any problem that you may encounter. If
you need further assistance, please do not hesitate to contact your local Biometra distributor or
Biometra.
Problem
Cause
Solution
Inaccurate positioning of
sealing
Check positioning of silicone sealing,
Electrophoresis
Leakage of gel cuvette
Dust on spacers
clean Spacer,
Acryl glide on spacers
do not apply Acryl Glide onto the
spacers
After heavy use this may
happen from time to time
Self adhesive slot forming units (024221) are included in the TGGE maxi
system
Support film is not properly
attached to the glass plate
Fix polybond film with adhesive tape
along the upper edge of the glass
plate
Block has been cleaned with
strong detergents or
aggressive chemicals
Contact Biometra
Amperage and Wattage
have been set to “0”
Set Amperage and Wattage to
maximum values (Electrophoresis
should be controlled by Voltage)
Coating of the thermoblock
is damaged, the safety
shutoff is activated
Contact Biometra
Wavelike migration front
Temperature inhomogeneity
under the gel due to excess
thermal coupling solution
Use as little as possible thermal
coupling solution (not more than 2ml)
No sigmoid melting curve
(perpendicular TGGE)
Fragment melts completely
Perform Poland analysis
Slotformer fall off
Acrylamide solution gets
behind the support film during
pouring the gel
Teflon film peels away from
the thermoblock
No current
Current oscillates
Gel interpretation
Optimize primer design
No separation of hetero duplex
Inappropriate fragment
samples
(parallel TGGE)
Wrong temperature gradient
Acrylamide of poor quality
Instruction Manual TGGE MAXI 05/2012
Perform Poland analysis
Perform perpendicular TGGE
Use only high quality chemicals (p.a.)
43
Irreproducible gels
Erratic temperature
distribution over and under
the gel
Use only minimum volume of thermal
coupling solution under the gel
Do not overlay gel with buffer
Silver staining
Bad silver stain
Strong background
Weak staining of bands
Chemicals of poor quality
Use only high quality chemicals
Stale water
Use only freshly prepared aqua bidest
Too much silver nitrate
Refer to the staining protocol
Insufficient washing after
incubation in staining
solution
Extend wash step, change water
frequently
Excessive washing after
binding of staining solution
Reduce wash step after staining
9.1 Optimization of parallel TGGE
To improve separation in parallel TGGE the gradient should start directly at the temperature
where the fragments start to melt (see perpendicular gel) and should be rather flat. This
means there should be only a moderate temperature increase over the whole gel. Different
fragments in one sample separate as soon as the first fragment starts to melt. At a certain
(higher) temperature the next fragment starts to melt. In a moderate gradient, the
temperature increase per centimeter is smaller than in a steeper gradient. This means, the
distance between two temperatures (i.e. locations in the gel) is bigger than in a steeper
temperature gradient. This results in a wider separation of fragments that melt at different
temperatures (see Figure 17).
A
3 0 °C
7 0 °C
B
3 0 °C
4 0 °C
44
Instruction Manual TGGE MAXI 05/2012
Figure 17: Parallel TGGE using a steep (A) or a flat (B) temperature gradient. With a smaller temperature gradient
(30 to 40 °C, B) the separation of samples is much wider. (Note: the temperatures in this figure are only for
demonstration.)
9.2 Optimization of TGGE for genetic fingerprinting of
microorganisms (mixed population analysis)
Primers should be designed thoroughly to avoid unspecific PCR products. Ideally sequences
of the species considered must be compared for identifying conserved regions 48. Further,
the PCR cycles should be reduced to a minimum 49 while extension times should be
enlarged 50 to avoid the formation of heteroduplex artefacts 52. In cases of mixed-template
PCR products a low cycler number reamplification may increase specificity 52. In general,
PCR products should be purified prior to TGGE analysis 51. Sometimes PCR artefacts are
co-amplified which can result in smears on TGGE gels. PCR products can be purified by
PCR purification kits or ideally each PCR-product is subjected to conventional agarose
electrophoresis. After electrophoretic separation, for each sample the desired band can be
cut from the gel and purified.
9.3 Optimisation of Heteroduplex analysis
9.3.1
Principle of heteroduplex analysis
If the difference in melting temperature between wildtype and mutant is very small,
heteroduplex analysis is a rewarding approach. Heteroduplex analysis makes it very easy to
distinguish between the wildtype and mutant form of a DNA fragment. The basic principle is
to mix each sample with an external standard. In most cases this standard is a PCR
fragment without mutations, for example amplified from the wild type. After mixing the
standard DNA fragment with the PCR fragment from the sample the mixture is heated and
subsequently slowly cooled down (for protocol see section 5.3).
a
a
A
a
A
a
a
A
A
a
A
A
denature
re-anneal
Figure 18: Principle of heteroduplex analysis.
The re-annealing of sample and standard results in 4 different DNA fragments. 1) The wildtype homoduplex (AA),
2) the mutant homoduplex (aa), 3) and 4) two different heteroduplices (Aa and aA). These heteroduplices carry at
least one mismatch (disturbed base pairing) and have a significant lower melting temperature than the
homoduplices.
This procedure results in a complete denaturing of both double stranded PCR fragments and
a subsequent re-annealing. If the sample is different from the standard, re-annealing leads to
4 different double stranded DNA fragments (see Figure 18): 1) the homoduplex of the
standard (wildtype AA), 2) the homoduplex of the sample (mutant aa),3) and 4) two
heteroduplices between standard and sample (Aa and aA). Due to the differences between
sample and standard these heteroduplices display mismatches in their base pairing in least
one position. Such mismatches have a strong impact on the melting behavior because the
Instruction Manual TGGE MAXI 05/2012
45
number of base pairs between the two strands is reduced. Therefore the heteroduplices can
be easily separated from the homoduplices using TGGE.
electrophoresis
The identification of the optimum temperature gradient for the separation of a heteroduplex
analysis is absolutely the same as for a single fragment. The separation of a heteroduplex
sample in a perpendicular TGGE results in 4 different melting curves. The 2 heteroduplices
have a lower melting temperature and denature at a lower temperature compared to the
homoduplices.
heteroduplex Aa
heteroduplex aA
homoduplex aa
homoduplex AA
cold
warm
Figure 19: Separation of a heteroduplex sample in perpendicular TGGE.
The temperature gradient can then be adapted in the same way as for a conventional sample
(see chapter 4.4). In parallel TGGE, the samples melt as they migrate along the temperature
gradient. The heteroduplices (with mismatch) melt at a lower temperature than the
homoduplices. Thus they open earlier in the partial single strand and are slowed down in the
gel matrix. The homoduplices migrate a longer distance as complete double strands and
start to melt at a higher temperature (i.e. later in respect to the temperature gradient).
Therefore the lower bands in parallel TGGE are the homoduplices, whereas the higher
bands are the heteroduplices.
Figure 20: Schematic drawing of a screening multiple samples in a parallel TGGE. Both homoduplices (AA, aa)
have a higher melting temperature and migrate further in the gel. The heteroduplices melt at a lower temperature
resulting in a slower migration.
9.3.2
Evaluation of a heteroduplex analysis
There are two possible states in heteroduplex analysis: 1) the sample is identical to the
standard (wildtype) 2) the sample is different from the wildtype. In the former case, the
denaturation / renaturation procedure results in one (the same) homoduplex. The
46
Instruction Manual TGGE MAXI 05/2012
subsequent separation in parallel TGGE shows only a single band. In the latter case,
denaturation / renaturation leads to the four different populations depicted in Figure 19.
Separation in parallel TGGE results in up to four different bands (see Figure 20). If the
temperature gradient has not been correctly optimized, or if separation time was to short,
there may as well only be two or three bands.
This makes heteroduplex analysis very easy to evaluate:
number of bands
result
one
sample is identical to the standard
no mutation
more than one (up to 4
bands)
sample is different form the standard
mutation
9.4 The TGGE Test kit
The TGGE test kit (024-050) was developed to get familiar with the TGGE system. It consists
of 3 different DNA samples:
•
a wild type sample (DNA fragment without mutation)
•
a mutant sample (DNA fragment that differs in on position from the wild type)
•
the heteroduplex sample (sample has been prepared as described in section 9.3.1)
The samples are separated in a 8% PAA gel with 8M Urea and a 1 x TAE buffer system (for
preparation of gel solution and buffer see section 17.2).
9.4.1
Perpendicular TGGE using the Biometra TGGE test kit
1) sample preparation
mix 100 µl heteroduplex sample with
100 µl loading buffer TAE (see section 17.2)
2) Load heteroduplex sample to the broad slot of a perpendicular gel
3) Let sample migrate into the gel with 350V for approx. 20 minutes
4) Cover gel with cover film, assemble buffer wicks, cover plate and safety lid
5) start run
Temperature gradient
30 to 70°C
Voltage:
300V
Run time:
4h
Instruction Manual TGGE MAXI 05/2012
47
6) silver stain gel
9.4.2
Parallel TGGE using the Biometra TGGE test kit
1) sample preparation:
mix 5µl sample (wildtype or mutant or heteroduplex) with
10µl running buffer (TAE) and add
15µl loading buffer TAE (see section 17.2)
2) Assemble electrophoresis unit, cover gel with cover film (beneath the slots), assemble
cover plate and safety lid
3) Load 5 µl of wildtype, mutant and heteroduplex samples
6) Let sample migrate into the gel with 400V for approx. 5 minutes
4) After pre-run, cover gel like described in section 6.2.2
5) start main run
Temperature gradient
30 to 60 °C
Voltage:
400V
Run time:
3h
4) silver stain gel
48
Instruction Manual TGGE MAXI 05/2012
10. Maintenance and repair
10.1 Cleaning and Maintenance
The TGGE system is built to operate for a long time without the need for periodical
maintenance. Nevertheless, occasionally cleaning of the air inlet may be necessary to
maintain the efficiency of the thermoblock. The inlet for the airflow is located at sides of the
themoblock. Be sure that the inlet is not clogged by dust or other materials. Dust can be
removed easily from the inlet with a conventional vacuum cleaner. Additionally, the housing
may be cleaned from time to time with a smooth cotton cloth. Do not use strong detergents,
abrasives or organic solvents for cleaning.
Important: Appropriate safety regulations must be observed when working with infectious or
pathogenic material.
10.2 Servicing and repair
The TGGE system contains no user serviceable parts. Do not open the housing instrument.
Service and repair may only be carried out by the Biometra Service department or otherwise
qualified technical personal.
10.3 Replacement of Spare Parts
Only original spare parts mentioned in these operating instructions are allowed.
Instruction Manual TGGE MAXI 05/2012
49
11. Accessories
11.1 Consumables and spare parts
TGGE MAXI Starter kit (Germany only)
for 25 gels, including glass plate 1 slot (024-228), glass plate with spacer, no
slot former (024-227), applicator strip silicone, 34 slots, 8µl (024-223), 2 cover
plates (024-221), 2 sealings (024-230), 25 cover films (024-232), 25 Polybond
films (024-234), 4 buffer wicks (024-216), sample AcrylGlide™ (211-319), 12
binder clamps (024-207)
024-204
TGGE MAXI Starter kit (International)
for 25 gels, including glass plate 1 slot (024-228), glass plate with spacer, no
slot former (024-227), applicator strip silicone, 34 slots, 8µl (024-223), 2 cover
plates (024-221), 2 sealings (024-230), 25 cover films (024-232), 25 Polybond
films (024-234), 4 buffer wicks (024-216),12 binder clamps (024-207)
024-294
TGGE Test kit
024-050
Test DNA for perpendicular and parallel test runs: wild type DNA (control),
mutant DNA, hetero duplex DNA, sample buffer.
TGGE MAXI buffer wicks, 100/pcs. (18 x 20 cm)
024-215
TGGE MAXI buffer wicks, reusable, 8 pcs.
024-217
TGGE MAXI glass plate without Spacer 23.5 x 23.5 cm
024-221
Self adhesive slot forming units (8 strips with 28 units á 5µl each for parallel
gels, 9 strips with one broad slot á 200µl each for perpendicular gels)
024-222
Applicator strips 240mm (3 pcs.), 43 slots á 8µl each
024-223
TGGE Maxi glass plate with spacers and no slot fomers for use with
applicator strips (024-223)
024-227
TGGE MAXI glass plate perpendicular, with spacer (1mm) and slot former (1
slot, 75 µl)
024-228
TGGE MAXI glass plate parallel, with spacer (1mm) and slot former (32 slots,
5 µl)
024-229
TGGE MAXI silicon sealing for casting of gels, 1mm
024-230
TGGE MAXI gel cover film 25/pkg
024-232
TGGE MAXI polybond film 25/pkg
024-234
TGGE MAXI polybond film 100/pkg
024-235
50
Instruction Manual TGGE MAXI 05/2012
12. Service
Should you have any problems with this unit, please contact our service department or your
local Biometra dealer:
Biometra GmbH
Service Department
Rudolf-Wissell-Straße 14 - 16
D-37079 Göttingen
Phone:++49 (0)5 51 50 68 6 - 10 or 12
Fax: ++49 (0)5 51 50 68 6 -11
e-mail: [email protected]
If you would like to send the unit back to us, please read the following return
instructions in chapter 12.1.
12.1 Instructions for return shipment
In case of an instrument failure that cannot be fixed by the procedures described in section 9
please proceed as follows:
•
Return only defective devices. For technical problems which are not definitively
recognisable as device faults please contact the Technical Service Department at
Biometra (Tel.: +49 551-50881-10/12, Fax: +49 551-50881-11, e-mail:
[email protected]).
•
Please contact our service department for providing a return authorization number
(RAN). This number has to be applied clearly visible to the outer box. Returns without
the RAN will not be accepted!
•
Important: Carefully clean all parts of the instrument of biologically dangerous, chemical or
radioactive contaminants. If an instrument is contaminated, Biometra will be forced to
refuse to accept the device. The sender of the repair order will be held liable for possible
losses resulting from insufficient decontamination of the device.
•
Please prepare written confirmation that the device is free from biologically dangerous and
radioactive contaminants. The declaration of decontamination (see section 13) must be
attached to the outside of the packaging.
•
Use the original packing material. If not available, contact Biometra or your local
distributor.
•
Label the outside of the box with “CAUTION! SENSITIVE ELECTRONIC INSTRUMENT!”
•
Please enclose a note which contains the following:
a)
Sender’s name and address,
b)
Name of a contact person for further inquiries with telephone number,
c)
Description of the fault, which also reveals during which procedures the fault
occurred, if possible
Instruction Manual TGGE MAXI 05/2012
51
13. Equipment Decontamination Certificate
To enable us to comply with german law (i.e. §71 StrlSchV, §17 GefStoffV and §19 ChemG)
and to avoid exposure to hazardous materials during handling or repair, please complete this
form, prior to the equipment leaving your laboratory.
COMPANY / INSTITUTE
__________________________________________________
ADDRESS
______________________________________________________________
PHONE NO
_________________________
E-MAIL
______________________________________________________________
EQUIPMENT
FAX NO_________________________
Model
______________
Serial No
______________
______________
______________
______________
______________
______________
______________
If on loan / evaluation Start Date: __________________
Finish Date __________________
Hazardous materials used with this equipment:
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
Method of cleaning / decontamination:
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
The equipment has been cleaned and decontaminated:
NAME __________________________________ POSITION _______________________
(HEAD OF DIV./ DEP./ INSTITUTE / COMPANY)
SIGNED ________________________________ DATE ____________________________
PLEASE RETURN THIS FORM TO BIOMETRA GMBH OR YOUR LOCAL BIOMETRA
DISTRIBUTOR TOGETHER WITH THE EQUIPMENT.
PLEASE ATTACH THIS CERTIFICATE OUTSIDE THE PACKAGING. INSTRUMENTS
WITHOUT THIS CERTIFICATE ATTACHED WILL BE RETURNED TO SENDER.
52
Instruction Manual TGGE MAXI 05/2012
General Information for Decontamination:
Please contact your responsible health & safety officer for details.
Use of radioactive substances:
Please contact your responsible person for details.
Use of genetically change organism or parts of those:
Please contact your responsible person for details.
Instruction Manual TGGE MAXI 05/2012
53
14. Note for the disposal of electric / electronic waste.
Note
for disposal of electric / electronic waste
Hinweis
für die Entsorgung von Elektroaltgeräten
Renseignement
du traitement des déchets des appareils
électrique / électronique
This symbol (the crossed-out wheelie bin) means, that this product should be brought to the return and / or
separate systems available to end-users according to yours country regulations, when this product has reached
the end of its lifetime.
For details, please contact your local distributor!
This symbol applies only to the countries within the EEA*.
EEA = European Economics Area, comprising all EU-members plus Norway, Iceland and Liechtenstein.
-------------------------------------------------------------------------------------------------------------------------------------------------------
Dieses Symbol (die durchgestrichene Abfalltonne) bedeutet, dass dieses Produkt von der Firma Biometra
für eine kostenlose Entsorgung zurückgenommen wird. Dies gilt nur für Geräte, die innerhalb
Deutschlands gekauft worden sind.
Kontaktieren Sie für die Entsorgung bitte die Biometra Service-Abteilung!
Außerhalb Deutschlands wenden Sie sich bitte an den lokalen Händler.
Dieses Symbol gilt nur in Staaten des EWR*.
*EWR = Europäischer Wirtschaftsraum, umfasst die EU-Mitgliedsstaaten sowie Norwegen, Island und
Liechtenstein.
------------------------------------------------------------------------------------------------------------------------------------------------------Cet symbol (conteneur à déchets barré d´une croix) signifie que le produit, en fin de vie, doit être retourné
à un des systèmes de collecte mis à la disposition des utilisateurs finaux en conséquence des
régulations par la loi de votre pays.
Pour des information additionel nous Vous demandons de contacter votre distributeur!
Cet symbole s´ápplique uniquement aux pays de l´EEE*.
EEE = Espace économique européen, qui regroupe les États membres de l´UE et la Norvège, Islande et le
Liechtenstein.
54
Instruction Manual TGGE MAXI 05/2012
15. Declaration of Conformity
EU – Konformitätserklärung
EC - Declaration of Conformity
Göttingen, 19. November 2008
im Sinne der EG-Richtlinie über elektrische Betriebsmittel zur Verwendung innerhalb
bestimmter Spannungsgrenzen 73/23/EWG
following the EC directive about electrical equipment for use within certain limits of voltage
73/23/EEC
und / and
im Sinne der EG-Richtlinie für die elektromagnetische Verträglichkeit 89/336/EWG.
following the EC directive about the electromagnetic compatibility 89/336/EEC .
Hiermit erklären wir, dass folgende Elektrophoresegeräte:
Herewith we declare that the following gel electrophoresis systems:
Typen / types:
TGGE MAXI System (German)
TGGE MAXI System (International)
TGGE MAXI Controller
TGGE MAXI Elektrophorese Kammer mit Temperierblock
TGGE MAXI Power Supply
TGGE MAXI system (German)
TGGE MAXI system (International)
TGGE MAXI controller
TGGE MAXI electrophoresis unit with Peltier element powered
gradient block
TGGE MAXI power supply
Best.-Nr. / Order No.:
024-200, 024-294, 024-201, 024-202, 024-203, 024-290
den grundlegenden Anforderungen der
corresponds to the basic requirements of
EG-Niederspannungsrichtlinie 73/23/EWG und der
EC low voltage directive 73/23/EEC and the
EG-Richtlinie über die elektromagnetische Verträglichkeit 89/336/EWG entsprechen.
EC directive about the electromagnetic compatibility 89/336/EEC .
Folgende harmonisierte Normen wurden angewandt:
The following harmonized standards have been used:
EN 55022 + A1 + A2
EN 61000-3-3 + A1
EN 61010-2-010 + A1
Dr. Jürgen Otte
Quality Manager
Instruction Manual TGGE MAXI 05/2012
55
Formular 006, Formular
Ver. 03 006, Ver. 03
EN 55011 + A1 + A2
EN 61000-3-2
EN 61000-6-1
EN 61010-1
16. Warranty
This Biometra instrument has been carefully build, inspected and quality controlled before
dispatch. Hereby Biometra warrants that this instrument conforms to the specifications given
in this manual. This warranty covers defects in materials or workmanship as described under
the following conditions:
This warranty is valid for 24 month from date of shipment to the customer from Biometra.
This warranty will not be extended to a third party without a written agreement of Biometra.
This warranty covers only the instrument and all original accessories delivered with the
instrument. This warranty is valid only if the instrument is operated as described in the
manual.
Biometra will repair or replace each part which is returned and found to be defective. This
warranty does not apply to wear from normal use, failure to follow operating instructions,
negligence or to parts altered or abused.
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Instruction Manual TGGE MAXI 05/2012
17. Appendix
17.1 References
1.
Riesner, D., Henco, K. and Steger, G. (1990): Temperature-Gradient Gel
Electrophoresis: A method for the analysis of conformational transitions and mutations
in nucleic acids and protein. Page 169-250 In Chrambach, A., Dunn, M.J., Radola,
B.J.: Advances in Electrophoresis, Vol. 4, VCH Verlagsgesellschaft Weinheim
2.
Kappes, S. et al.(1995): p53 mutations in ovarian tumors, detected by temperaturegradient gel electrophoresis, direct sequencing and immunohistochemistry. Int. J.
Cancer 64: 52-59
3.
Milde-Langosch, K. et al. (1995): Presence and persistence of HPV and p53 mutation
in cancer of the cervix uteri and the vulva. Int. J. Cancer 63: 639-645
4.
Horn, D. et al.(1996): Three novel mutations of the NF1 gene detected by temperature
gradient gel electrophoresis of exons 5 and 8. Electrophoresis 17: 1559-1563
5.
Wieland, U. et al.(1996): Quantification of HIV-1 proviral DNA and analysis of genomic
diversity b ypolymerase chain reaction and temperature gradient gel electrophoresis. J.
Virology Methods 57: 127-139
6.
Kuhn, J.E. et al. (1995): Quantitation of human cytomegalovirus genomes in the brain
of AIDS patients. Journal of Medical Virology 47: 70-82
7.
Linke, B. et. al. (1995): Identification and structural analysis of rearranged
immunoglobulin heavy chain genes in lymphomas and leukemia. Leukemia 9: 840847.
8.
Menke M.A. et al. (1995): Temperature gradient gel electrophoresis for analysis of a
polymerase chain reaction-based diagnostic clonality assay in the early stages of
cutaneous T-cell lymphomas.
9.
Hecker, R. et al. (1988): Analysis of RNA structure by temperature-gradient gel
electrophoresis: viroid replication and processing. Gene 72: 59-74
10.
Baumstark, T. and Riesner, D. (1995): Only one of four possible secondary structures
of the central conserved region of potato spindle tuber viroid is a substrate for
processing in a potato nuclear extract. Nucleid Acids Research 23: 4246-4254
11.
Loss, P., Schmitz, M., Steger, G. and Riesner, D. (1991): Formation of a
thermodynamically metastable structure containing hairpin II is critical for the potato
spindle tuber viroid. EMBO Journal 10: 719-728
12.
Riesner, D. (1998): Nucleic acid structures. In: Antisense Technology. Practical
Approach Series. Oxford University Press. p1-24 (in press)
13.
Wiese, U. et al. (1995): Scanning for mutations in the human prion protein open
reading frame by temporal temperature gradient gel electrophoresis. Electrophoresis
16: 1851-1860
14.
Nubel, U. et al. (1996): Sequence heterogenities of genes encoding 16S rRNAs in
Paenibacillus polymyxa detected by temperature gradient gel electrophoresis.
Instruction Manual TGGE MAXI 05/2012
57
15.
Lessa, E.P. and Applebaum, G. (1993): Screening techniques for detecting allelic
variation in DNA sequences. Molecular Ecology 2: 119-129
16.
Richter, A., Plobner, L., Schumacher, J. 1997: Quantitatives PCR-Verfahren zur
Bestimmung der Plasmidkopienzahl in rekombinanten Expressionssystemen.
BIOforum 20: 545-547
17.
Henco, K. and Heibey, M. (1990): Quantitative PCR – the determination of template
copy numbers by temperature gradient gel electrophoresis. Nucleic Acids Research
18: 6733-6734
18.
Birmes, A. et al. (1990): Analysis of the conformational transition of proteins by
temperature-gradient gel electrophoresis. Electrophoresis 11: 795-801
19.
Arakawa, T. et al. (1993): Analysis of the heat-induced denaturation of proteins using
temperature gradient gel electrophoresis. Analytical Biochemistry 208: 255-259
20.
Chen, X. et al. (1995): High resolution SSCP by optimization of the temperature by
transverse TGGE. Nucleic Acids Research 23: 4524-4525
21.
Scholz, R.B. et al. (1993): Rapid screening for Tp53 mutations by temperature gradient
gel electrophoresis: a comparison with SSCP analysis. Human Molecular Genetics 2:
2155-2158
22.
Elphinstone and Baverstock, P.R. (1997): Detecting mitochondrial genotypes by
temperature gradient gel electrophoresis and heteroduplex analysis. BioTechniques
23: 982-986
23.
Poland, D. (1974): Recursion relation generation of probability profiles for sequencespecific macromolecules with long-range correlations. Biopolymers 13:1859-1871
24.
Lerman, L.S. and Silverstein, K. (1987): Computational simulation of DNA-melting and
its application to denaturing gradient-gel electrophoresis. Meth. Enzymol. 155: 482-501
25.
Steger, G. (1994): Thermal denaturation of double-stranded nucleic acids: prediction of
temperatures critical for gradient electrophoresis and polymerase chain reaction.
26.
Schumacher, J, Randels, J.W. and Riesner,D. (1983): A two dimensional
electrophoretic technique for detection of circular viroids and virusoids. Anal. Biochem.
135, 288 - 295
27.
Sambrook, J., Fritsch, E.F. and Maniatis,T. (1989): Molecular cloning, Cold Spring
Habor Laboratory press
28.
Steger, G. and Riesner, D. (1992): Temperaturgradienten-Gelelektrophorese: eine
Methode zur Analyse von Konformationsübergängen und Mutationen in Nukleinsäuren
und Proteinen. In Radola, B.J. (ed) Handbuch der elektrophorese, VCH
Verlagsgesellschaft, Weinheim
29.
Sheffield, V.C., Cox, D.R. and Lerman, R.M. (1989): Attachment of a 40-base-pair
G+C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain
reactiob results in improved detection of single-base changes. Proc. Natl. Acad. Sci.
USA 86, 232 - 236
58
Instruction Manual TGGE MAXI 05/2012
30.
Hecker R., Wang Z., Steger G. and Riesner D. (1988): Analysis of RNA structure by
temperature-gradient gel electrophoresis: viroid replication and processing. Gene 72,
59-74
31.
Jiang L., Chen W., Tain L.P. and Liu Y. (1991): Temperature-gradient gel
electrophoresis of apple scar skin viroid. Acta Microbiol. Sin. 30, 278-283
32.
Riesner D., Hecker R. and Steger G. (1988): Structure of viroid replication
intermediates as studied by thermodynamics and temperature-gradient gel
electrophoresis. In Sarma R.H. and Sarma M.H. (eds.) Structure & Expression, Vol. I:
From Proteins to Ribosomes, Adenine press, 261-285
33.
Riesner D., Steger G., Zimmat R., Owens R.A., Wagenhöfer M., Hillen W., Vollbach S.
and Henco K. (1989): Temperature-gradient gel electrophoresis of nuleic acids:
Analysis of confor-mational transitions, sequence variations, and protein-nucleic acid
interactions. Electrophoresis 10, 377-389
34.
Rosenbaum V. and Riesner D. (1987): Temperature-gradient gel electrophoresis:
thermodynamic analysis of nucleic acids and proteins in purified form and in cellular
extract. Biohys. Chem. 26, 235-246
35.
Schönborn J., Oberstraß J., Breyel E., Tittgen J., Schumacher J., Lukacs N. (1991):
Monoclonall antibodies to double-stranded RNA as probes of RNA structure in crude
nucleic acid extracts. Nucleic Acids Res. 19, 2993-3000
36.
Po Tien, Steger G., Rosenbaum V., Kaper J. and Riesner D. (1987): Double-stranded
cucumovirus associated RNA5: experimental analysis of nec-rogenic and nonnecrogenic variants by temperature-gradient gel electrophoresis. Nucleic Acids Res.
15, 5069-5083
37.
Zimmat R., Gruner R., Hecker R., Steger G. and Riesner D. (1991): Analysis of
mutations in viroid RNA by non-denaturing and temperature-gradient gel
electrophoresis. In R.H. Sarma and M.H. Sarma (eds.) Structure & Methods, Vol. 3:,
DNA & RNA, Adenine Press, 339-357
38.
Rosenbaum V., Klahn T., Lundberg Holmgren E., von Gabain A. and Riesner D.
(1992): Co-existing structures of an mRNA stability determinat: The 5‘ region of the
Escherichia coli and Serratia marcescens ompA mRNA, J.Mol.Biol., in press
39.
Birmes A., Sättler A., Maurer S.O. and Riesner D. (1990): „Analysis of the
conformational transitions of proteins by temperature-gradient gel electrophoresis“.
Electrophoresis 11, 795-801
40.
Sättler A., Kanka S., Schrörs W. and Riesner D. (1992): „Random mutagenesis of the
weak calcium binding side in SubtilisinCarlsberg and screening for thermal stability by
temperature-gradient gel electrophoresis“. Accepted for: 1st International Symposium
of Subtilisin Enzymes, EMBL, Hamburg
41.
Thatcher D. and Hodson B. (1981): „Denaturation of proteins and nucleic acids by
thermal-gradient electrophoresis“. Biochem. J. 197, 105-109
42.
Wagenhöfer M., Hansen D. and Hillen W. (1988): „Thermal denaturation of engineered
tet repressor proteins and their complexes with tet operator and tetracycline studie by
temperature-gradient gel electrophoresis“. Analytical Biochem. 175, 422-432
43.
Sanguinetti C.J., Neto E.D. and Simpson A.J.G. (1994): BioTechniques 17, 915
Instruction Manual TGGE MAXI 05/2012
59
44.
Kappes, S., Milde-Langosch, K., Kressin, P., Passlack, B., Dockhorn-Dwornczak,B.,
Röhlke, P. and Löning, T. (1995): "p53 Mutations in ovarian tumors, detected by
temperature-gradient gel electrophoresis, direct sequencing and
immunohistochemistry". Int. J. Cancer 64, 52 - 59
45.
Kluwe, L., MacCollin, M., Tatagiba, M., Thomas, S., Hazim, W., Haase, W. and
Mautner, V.-F. (1998): "Phenotypic variability associated with 14 splice-site mutations
in the NF2 gene". American Journal of Medical Genetics 77, 228 - 233
46.
Lerman, L. S. and Beldjord, C. (1998). "Comprehensive mutation detection with
denaturing gradient gel electrophoresis". In R.G.H. Cotton, E. Edkins and S. Forrest
(eds.) Mutation Detection, A Practical Approach, Oxford University Press, 35 - 62
47.
Gamper, H., Piette, J. and Hearst, J.E. (1984): Efficient formation of a crosslinkable
HMT monoadduct at the Kpn I recognition site. Photochem. Photobiol. 40, 29 ff
48.
Smit, E., Leeflang, P., Glandorf, B., van Elsas, J.D. and Wernars, K. (1999): Analysis
of fungal diversity in the wheat rhizosphere by sequencing of cloned PCR-amplified
genes encoding 18S rRNA and temperature gradient gel electrophoresis. Appl.
Environ. Microbiol. 65(6), 2614-2621
49.
Qiu X, Wu L, Huang H, McDonel PE, Palumbo AV, Tiedje JM, Zhou J (2001):
Evaluation of PCR-generated chimeras, mutations, and heteroduplexes with 16S rRNA
gene-based cloning. Appl Environ Microbiol. Feb; 67(2):880-7.
50.
Jumpponen A. (2007): Soil fungal communities underneath willow canopies on a
primary successional glacier forefront: rDNA sequence results can be affected by
primer selection and chimeric data. Microb Ecol. Feb; 53(2):233-46.
51.
He J, Xu Z, Hughes J. (2006): Molecular bacterial diversity of a forest soil under
residue management regimes in subtropical Australia. FEMS Microbiol Ecol. Jan;
55(1):38-47. Appendix
52.
Thompson JR, Marcelino LA, Polz MF. (2002): Heteroduplexes in mixed-template
amplifications: formation, consequence and elimination by 'reconditioning PCR'.
Nucleic Acids Res. May 1; 30(9):2083-8.
53.
Trabal N, Mazón-Suástegui JM, Vázquez-Juárez R, Asencio-Valle F, MoralesBojórquez E, Romero J. Molecular Analysis of Bacterial Microbiota Associated with
Oysters (Crassostrea gigas and Crassostrea corteziensis) in Different Growth Phases
at Two Cultivation Sites. Microb Ecol. 2012 Mar 27.
54.
Rojas-Oropeza M, Fernández FJ, Dendooven L, Cabirol N. Effect of methyl parathion
on nitrous oxide production: a laboratory study. J Environ Manage. 2012 Mar; 95
Suppl:S25-30
55.
Sun L, Ouyang X, Tang Y, Yang Y, Luo Y. Effects of different methods of DNA
extraction for activated sludge on the subsequent analysis of bacterial community
profiles. Water Environ Res. 2012 Feb;84(2):108-14
56.
Wang X, Song M, Wang Y, Gao C, Zhang Q, Chu X, Fang H, Yu Y. Response of soil
bacterial community to repeated applications of carbendazim. Ecotoxicol Environ Saf.
2012 Jan;75(1):33-9
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Instruction Manual TGGE MAXI 05/2012
57.
Purswani J, Martín-Platero AM, Reboleiro-Rivas P, González-López J, Pozo C.
Comparative analysis of microbial DNA extraction protocols for groundwater samples.
Anal Biochem. 2011 Sep 15;416(2):240-2
58.
Eusébio A, Tacão M, Chaves S, Tenreiro R, Almeida-Vara E. Molecular assessment of
microbiota structure and dynamics along mixed olive oil and winery wastewaters
biotreatment. Biodegradation. 2011 Jul;22(4):773-95
59.
Vílchez R, Gómez-Silván C, Purswani J, González-López J, Rodelas B.
Characterization of bacterial communities exposed to Cr(III) and Pb(II) in submerged
fixed-bed biofilms for groundwater treatment. Ecotoxicology. 2011 Jun;20(4):779-92
60.
Jeon BY, Jung IL, Park DH. Enrichment of CO2-fixing bacteria in cylinder-type
electrochemical bioreactor with built-in anode compartment. J Microbiol Biotechnol.
2011 Jun;21(6):590-8
61.
Poniková S, Tlučková K, Antalík M, Víglaský V, Hianik T. The circular dichroism and
differential scanning calorimetry study of the properties of DNA aptamer dimers.
Biophys Chem. 2011 Apr;155(1):29-35
62.
Calderón K, Rodelas B, Cabirol N, González-López J, Noyola A. Analysis of microbial
communities developed on the fouling layers of a membrane-coupled anaerobic
bioreactor applied to wastewater treatment. Bioresour Technol. 2011 Apr;102(7):461827
63.
Guernaoui S, Garcia D, Gazanion E, Ouhdouch Y, Boumezzough A, Pesson B,
Fontenille D, Sereno D. Bacterial flora as indicated by PCR-temperature gradient gel
electrophoresis (TGGE) of 16S rDNA gene fragments from isolated guts of
phlebotomine sand flies (Diptera: Psychodidae). J Vector Ecol. 2011 Mar;36 Suppl
1:S144-7
64.
de la Rua A, Rodelas B, González-López J, Gómez MA. Submerged filter biofilm
formation by nitrate-contaminated groundwater microbiota. J Environ Sci Health A Tox
Hazard Subst Environ Eng. 2011;46(10):1113-21
65.
Ahmed S, Komori M, Tsuji-Ueno S, Suzuki M, Kosaku A, Miyamoto K, Nishigaki K.
Genome profiling (GP) method based classification of insects: congruence with that of
classical phenotype-based one. PLoS One. 2011;6(8): e23963
66.
Seo H, Jeon BY, Yun A, Park DH. Effect of glasswort (Salicornia herbacea L.) on
microbial community variations in the vinegar-making process and vinegar
characteristics. J Microbiol Biotechnol. 2010 Sep;20(9):1322-30
67.
Lee WJ, Lee JK, Chung J, Cho YJ, Park DH. Effects of electrochemical reduction
reactions on the biodegradation of recalcitrant organic compounds (ROCs) and
bacterial community diversity. J Microbiol Biotechnol. 2010 Aug;20(8):1230-9
68.
Biasucci G, Rubini M, Riboni S, Morelli L, Bessi E, Retetangos C. Mode of delivery
affects the bacterial community in the newborn gut. Early Hum Dev. 2010 Jul;86 Suppl
1:13-5
69.
Martins M, Faleiro ML, Chaves S, Tenreiro R, Costa MC. Effect of uranium (VI)on two
sulphate-reducing bacteria cultures from a uranium mine site. Sci Total Environ. 2010
May 15;408(12):2621-8
Instruction Manual TGGE MAXI 05/2012
61
70.
Ecke TH, Schlechte HH, Schiemenz K, Sachs MD, Lenk SV, Rudolph BD, Loening SA.
TP53 gene mutations in prostate cancer progression. Anticancer Res. 2010
May;30(5):1579-86
71.
Martins M, Faleiro ML, Chaves S, Tenreiro R, Santos E, Costa MC. Anaerobic bioremoval of uranium (VI) and chromium (VI): comparison of microbial community
structure. J Hazard Mater. 2010 Apr 15;176(1-3):1065-72
72.
Gómez-Silván C, Molina-Muñoz M, Poyatos JM, Ramos A, Hontoria E, Rodelas B,
González-López J. Structure of archaeal communities in membrane-bioreactor and
submerged-biofilter wastewater treatment plants. Bioresour Technol. 2010
Apr;101(7):2096-105
73.
Sánchez-Peinado Mdel M, González-López J, Martínez-Toledo MV, Pozo C, Rodelas
B. Influence of linear alkylbenzene sulfonate (LAS) on the structure of
Alphaproteobacteria, Actinobacteria, and Acidobacteria communities in a soil
microcosm. Environ Sci Pollut Res Int. 2010 Mar;17(3):779-90
74.
Jeon BY, Seo HN, Kang SW, Park DH. Effect of electrochemical redox reaction on
biochemical ammonium oxidation and chemical nitrite oxidation. J Microbiol Biotechnol.
2010 Mar;20(3):485-93
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Instruction Manual TGGE MAXI 05/2012
17.2 Buffers
17.2.1 Running buffers:
TBE Running buffer 0.1 x conc. TBE (up to 1 x conc. TBE is possible)
10 x TBE (stock solution)
890 mM Boric Acid
20 mM EDTA
890 mM TRIS
Do not titrate to adjust pH!
TAE Running Buffer 1 x conc. TAE, pH 8.0
50 x TAE (stock solution)
(10
conc.)
pH x8.0
MOPS-Running Buffer
50x MOPS (stock solution)
MOPS/EDTA (ME)
(50 x conc.)
242g Tris base (2M)
57,1 ml glacial acid
100ml 0.5M EDTA (pH 8.0)
1 x conc. MOPS
1M MOPS
50 mM EDTA
pH = 8.0
17.2.2 Loading buffers:
Loading buffer TBE
TBE running buffer
0.1% Triton-X 100
0.01% Bromophenol Blue dye
0.01% Xylene Cyanol dye
Loading buffer TAE
Instruction Manual TGGE MAXI 05/2012
TAE running buffer
0.1% Triton-X 100
0.01% Bromophenol Blue dye
0.01% Xylene Cyanol dye
2 mM EDTA
63
Loading buffer MOPS
MOPS running buffer
1 mM EDTA
0.05% Bromophenol Blue dye
0.05% Xylene Cyanol dye
pH = 8.0
17.3 Other buffers:
TE buffer
10 mM Tris/HCl
0.1 mM EDTA
pH = 8.0
TEMED
Solution of N,N,N’,N’tetramethylethylendiamine
APS
10% Ammonium persulfate
Glycerol 40%
40% glycerol in water
Glycerol 50%
50% glycerol in water
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Instruction Manual TGGE MAXI 05/2012
18. Subject index
Accessories ........................................... 50
Autoradiography .................................... 42
Buffer
Loading buffers.................................. 63
Buffers................................................... 63
Running buffers ................................. 63
Declaration of Conformity ...................... 55
Decontamination Certificate .................. 52
Disposal ................................................ 54
DNA
Denaturation ...................................... 22
Elution ............................................... 42
Renaturation ...................................... 22
Electrophoresis
Setup ................................................. 28
GC clamps ............................................ 18
Gel
casting ............................................... 23
Gel solution ....................................... 25
pouring .............................................. 27
Installation ............................................. 13
Legal Notes ........................................... 10
Maintenance.......................................... 49
Operation Voltage ................................. 14
Poland analysis ..................................... 16
Program
Print program..................................... 38
Select language................................. 38
Set block type .................................... 38
Program
create ................................................ 33
edit .................................................... 33
enter electrophoresis paramter .......... 35
enter temperatures ............................ 34
Instruction Manual TGGE MAXI 05/2012
name ................................................. 34
pause ................................................ 36
select ................................................ 33
start ................................................... 36
stop ................................................... 36
view temperatures ............................. 37
Programming ........................................ 33
Psoralen ............................................... 19
References ........................................... 57
Repair ................................................... 49
Return shipment ................................... 51
Safety and Warning Notices .................. 11
Sample preparation............................... 22
Service .................................................. 51
Servicing ............................................... 49
Silver staining ....................................... 41
SSCP primers ....................................... 19
Staining
Ethiudium bromide ............................ 42
Silver staining.................................... 41
Technical specifications .......................... 9
Temperature gradient ........................... 20
TGGE
genetic fingerprinting ....................26, 45
Heteroduplex analysis ....................... 45
optimisation ..................................15, 16
parallel ............................... 7, 31, 44, 48
perpendicular ............... 7, 20, 30, 31, 47
TGGE controller
Programming .................................... 33
TGGE test kit ...................................47, 48
TGGE Test kit ....................................... 47
Trouble shooting ................................... 43
Warranty ............................................... 56
65
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