TGGE Maxi User Manual

TGGE Maxi User Manual
__________________________________ 1
TGGE MAXI System
(order number 024-200)
Manual
Version 3.02
October 2000
!! Warning !!
Please read this manual carefully before
using the apparatus
Biometra
biomedizinische Analytik GmbH
Rudolf-Wissell-Str. 30, D-37079 Goettingen
Tel.: 0551/50 686-0; Fax: 0551/50 686-66
email: [email protected]
http://www.biometra.com
__________________________________ I
1.
WHAT’S NEW IN THIS MANUAL .......................................................................................................... 2
1.1
2.
SAFETY INSTRUCTIONS / GENERAL REMARKS............................................................................................. 4
INTRODUCTION........................................................................................................................................ 5
2.1
2.2
PRINCIPLE OF THE METHOD ........................................................................................................................ 5
SPECIAL FEATURES OF THE BIOMETRA TGGE SYSTEM ............................................................................. 6
3.
BEFORE YOU START: THE TGGE FLOW CHART OF SUCCESS .................................................. 7
4.
THE TGGE MAXI SYSTEM ..................................................................................................................... 9
4.1
4.2
4.3
5.
SYSTEM OVERVIEW .................................................................................................................................... 9
INSTALLATION ........................................................................................................................................... 9
ADAPTATION OF PROTOCOLS FROM THE TGGE “MINI” SYSTEM ................................................................ 9
CASTING OF GELS ................................................................................................................................. 10
5.1 ASSEMBLY OF THE GEL CUVETTE ............................................................................................................. 10
5.2 PREPARING GEL SOLUTION ....................................................................................................................... 12
5.2.1
Preparing gel solution for TBE buffer system ............................................................................... 12
5.2.2
Preparing gel solution for MOPS / EDTA buffer system............................................................... 12
5.3 POURING GELS ......................................................................................................................................... 13
6.
ELECTROPHORESIS.............................................................................................................................. 14
6.1 ELECTROPHORESIS CONDITIONS............................................................................................................... 14
6.2 PRE-RUN FOR SAMPLE LOADING AND TEMPERATURE EQUILIBRATION...................................................... 14
6.3 SETUP ELECTROPHORESIS UNIT ................................................................................................................ 15
6.3.1
Prepare prior to assembly of the electrophoresis unit................................................................... 15
6.3.2
Gel setup for electrophoresis......................................................................................................... 17
6.4 PERPENDICULAR TGGE........................................................................................................................... 18
6.5 PARALLEL TGGE .................................................................................................................................... 19
6.6 HOW TO IDENTIFY THE OPTIMUM TEMPERATURE RANGE FROM A PERPENDICULAR GEL ........................... 19
7.
PROGRAMMING THE TGGE CONTROLLER .................................................................................. 21
7.1 CREATE / EDIT PROGRAM ......................................................................................................................... 21
7.2 SELECT PROGRAM .................................................................................................................................... 21
7.3 NAME PROGRAM ...................................................................................................................................... 22
7.4 ENTER TEMPERATURES FOR THE GRADIENT BLOCK.................................................................................. 22
7.5 ENTER ELECTROPHORESIS PARAMETERS .................................................................................................. 23
7.6 START ELECTROPHORESIS ........................................................................................................................ 24
7.7 STOP/PAUSE ELECTROPHORESIS ............................................................................................................... 25
7.8 VIEW TEMPERATURES OF THE GRADIENT ................................................................................................. 25
7.9 SPECIAL FUNCTIONS................................................................................................................................. 26
7.9.1
Print programs .............................................................................................................................. 26
7.9.2
Select / de-select signal.................................................................................................................. 26
7.9.3
Select language.............................................................................................................................. 26
7.9.4
Set block type................................................................................................................................. 26
8.
STAINING.................................................................................................................................................. 28
8.1 SILVER STAINING ..................................................................................................................................... 28
8.1.1
Silver staining protocol ................................................................................................................. 28
8.2 ETHIDIUM BROMIDE-STAINING................................................................................................................. 29
8.3 AUTORADIOGRAPHY ................................................................................................................................ 29
8.4 ELUTION OF DNA FROM THE TGGE GEL................................................................................................. 30
9.
SAMPLE PREPARATION....................................................................................................................... 31
9.1.1
9.1.2
9.1.3
10.
Purity of samples ........................................................................................................................... 31
Sample preparation for direct DNA analysis................................................................................. 31
Denaturation / Renaturation for heteroduplex analysis of DNA ................................................... 31
OPTIMIZATION OF TGGE.................................................................................................................... 32
II________________________________________________________ TGGE MAXI manual
10.1
DESIGN OF DNA FRAGMENT FOR TGGE ............................................................................................ 32
10.1.1 Poland analysis ............................................................................................................................. 32
10.1.2 GC clamps ..................................................................................................................................... 35
10.1.3 Chemical clamp with Psoralen (Furo[3,2-g]coumarin, C11H6O3)................................................. 35
10.1.4 Use of SSCP primers .................................................................................................................... 35
10.2
FIND CORRECT TEMPERATURE GRADIENT............................................................................................ 35
10.3
PARALLEL ANALYSIS OF MULTIPLE SAMPLES ...................................................................................... 36
10.4
OPTIMIZATION OF PARALLEL TGGE .................................................................................................. 37
10.5
WHAT TO DO, IF DIFFERENT SAMPLES HAVE VERY SIMILAR MELTING POINTS: HETERODUPLEX
ANALYSIS WITH TGGE...................................................................................................................................... 38
10.5.1 Principle of heteroduplex analysis ................................................................................................ 38
10.5.2 Evaluation of a heteroduplex analysis........................................................................................... 40
11.
THE TGGE TEST KIT (ORDER NUMBER 024-050) .......................................................................... 41
11.1
11.2
PERPENDICULAR TGGE USING THE BIOMETRA TGGE TEST KIT ........................................................ 41
PARALLEL TGGE USING THE BIOMETRA TGGE TEST KIT .................................................................. 42
12.
TECHNICAL SPECIFICATION............................................................................................................. 43
13.
ORDERING INFORMATION:................................................................................................................ 44
14.
TROUBLE-SHOOTING ........................................................................................................................... 45
15.
REFERENCES........................................................................................................................................... 47
16.
APPENDIX................................................................................................................................................. 50
16.1
BUFFERS ............................................................................................................................................. 50
17.
INSTRUCTIONS FOR RETURN SHIPMENT...................................................................................... 52
18.
EQUIPMENT DECONTAMINATION CERTIFICATE ...................................................................... 53
19.
WARRANTY ............................................................................................................................................. 54
2 ________________________________________________________ TGGE MAXI manual
1. What’s new in this manual
The current version of the TGGE maxi manual reflects the strong progress that has been made
with the TGGE method. New insights concerning handling and separation conditions have
been integrated in the proven step by step protocol.
The major news are:
1) The volume of thermal coupling solution is a critical factor for an even migration front
and should be held as small as possible (section 6.3.2)
2) Overlaying the gel with buffer should be omitted (section 6.3.2)
3) The gel cover film should be applied right after putting the gel on the block (section 6.3.2)
4) Identical salt concentration in all samples is important for an even migration front.
5) Instead of buffer wicks, household sponges may be used (section 6.3.2)
6) Extended Silver staining protocol for optimum gels
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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-Glide is a trademark of Amresco Inc.
Biometra is a trademark of Biometra GmbH.
Whatman is a trademark of Whatman International Ltd.
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.
Please read the TGGE MAXI manual carefully before starting operation
4 ________________________________________________________ TGGE MAXI manual
1.1 Safety instructions / general remarks
•
Do not fill buffer chambers above marking for maximum level
•
If buffer has been spilled on the electrophoresis unit, clean it carefully before start of
electrophoresis
•
Never run device without gel cover plate
•
The thermoblock is covered with a Teflon film. Avoid damaging this film.
•
For cleaning of the thermoblock do not use aggressive chemicals or strong detergents.
•
Do not use paraffin oil on the thermoblock.
•
In case of strong condensation under the safety lid stop run, dry instrument and re-start
•
Switch off power before removing the safety lid
•
Do not move instrument during operation
•
Do not lift the electrophoresis unit by holding it on the white frame. Instead, lift the
lower part of the device (red corpus).
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2. 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. Oncology2-4, Virology5,6, Immunology7,8, RNA Viroid
Research9-12, Prion Research13, Population Analysis14-15. The TGGE method has also been
used for quantitative analysis in industry16-17 and for conformational analysis of proteins18-19.
2.1
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.
6 ________________________________________________________ TGGE MAXI manual
2.2 Special features of the Biometra TGGE System
The most powerful characteristic of the Biometra TGGE system is the high reproducibility of
the temperature gradient. In contrast to conventional systems using chemical gradients
(DGGE) the temperature gradient of the Biometra TGGE system establishes the same
denaturing gradient time after time after time. The microprocessor driven gradient block of
the TGGE System allows strictly defined linear temperature gradients with high resolution
and reproducibility.
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 evident20-22.
The Biometra TGGE system is available in two formats. The standard TGGE “mini” system
(024-000) operates small gels and is therefore ideally suited for fast, serial experiments. The
TGGE maxi system (024-200) 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
Figure 2: Typical results after perpendicular TGGE (A: temperature gradient from left to
right) and parallel TGGE (B: temperature gradient from top to bottom).
__________________________________ 7
3. Before you start: The TGGE flow chart of success
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 10, Optimization of TGGE analysis.
Step 1 Check your DNA sequence 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.
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.
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, if the
perpendicular gel does not show a defined
melting curve.
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.
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4. The TGGE maxi system
4.1 System overview
The TGGE MAXI system consists of three components:
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
4.2 Installation
Connect electrophoresis unit and controller.
Connect controller and power supply
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.
4.3 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 10.2 and 10.3.
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 6.6).
10 _______________________________________________________ TGGE MAXI manual
5. Casting of gels
5.1 Assembly of the gel cuvette
There are two kind of gels for parallel TGGE analysis. The standard gel contains 30 slots for
5µl of sample each. This system is described in the bellow text.
Alternatively, a gel without slots can be casted (use glass plate wihout slot formers, 024-227).
In this case, samples are loaded with an applicator strip (024-223). 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.
The standard gel cuvette consists of one glass plate with spacers and slot formers (024-228 or
024-229), cover plate without spacers (024-221) and a silicone sealing (024-230). The
sandwich is fixed with 9 metal clamps.
Note: The gel is poured on a support film to optimize temperature transition between block
and gel. The gel is bound covalently to this hydrophilic film (024-234).
Note: The gels sticks to the support film throughout the whole procedure, including staining.
1)
Clean glass plates with 70% ethanol and a soft tissue.
Note : Be careful not to damage the slot formers when cleaning the glass plates. Never use
strong detergents in the area of the slot formers. Avoid strong mechanical contact. Self
adhesive slot forming units for replacement (024-222) are included in the starter kit.
_________________________________ 11
2)
Pre-treatment of glass plate with spacers and slot formers:
The glass plates with the slot formers are treated with a solution that makes the surface
hydrophobic and therefore facilitates the removal of the gel (the gel remains on the
support film)
* Apply approx. 2ml AcrylGlide (211-319) on the glass plate and spread it with a soft
tissue, especially between the slot formers (alternative hydrophobic solutions may be
used)
* wait for 2 minutes
* polish glass plate with a soft tissue
Note: Never apply Acryl Glide onto the spacers, because this will lead to leakage of the gel
cuvette.
3)
Place polybond film (024-234) on the glass plate without spacers.
4)
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 to the glass plate.
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.
5)
Place the silicone sealing around spacers.
6)
Assemble gel sandwich and fix it with 3 metal clamps on each side.
Note: the clamps should be placed directly on the spacers.
7)
Set gel sandwich upright on the 3 bottom clamps (see Figure 3)
12 _______________________________________________________ TGGE MAXI manual
Figure 3: Final setup of the gel cuvette.
5.2 Preparing gel solution
The choice of the buffer system has a strong impact on TGGE analysis. Concentration of salt
and denaturing agents (urea or formatted) 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 favorable, 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. In the following, two standard protocols for TBE and MOPS buffer systems are listed.
Please note, that the buffer system should be adapted to each special kind of application.
5.2.1
Preparing gel solution for TBE buffer system
For one TGGE maxi gel prepare 50 ml gel solution
gel composition
final concentrations
Acrylamid [8%]
urea [7M]
TBE [0.1x]
Glycerol [2%]
adjust with aqua bidest
STOCK SOLUTION
40 % (37,5 :1)
solid
1x
40%
FOR 50 ML
FOR 100 ML
10 ml
21 g
5 ml
2,5 ml
to 50 ml
20ml
42 g
10 ml
5 ml
To 100ml
•
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
TEMED
10 %
100%
80 µl
110 µl
160 µl
220 µl
•
Load gel solution in a syringe and attach a 0,4µm or 0,25µm sterile filter
•
Pour gel through sterile filter into the glas sandwich.
5.2.2
Preparing gel solution for MOPS / EDTA buffer system
Preparation of buffers and stock solutions is described in section 16.1.
For one TGGE maxi gel prepare 50 ml gel solution
_________________________________ 13
gel composition
final concentrations
STOCK SOLUTION
FOR 50 ML
FOR 100 ML
40 % (37,5 :1)
solid
50 x
40%
10 ml
21 g
1 ml
2,5 ml
to 50 ml
20ml
42 g
2 ml
5 ml
to 100ml
Acrylamid [8%]
urea [7M]
MOPS [1 x]
Glycerol [2%]
fill up 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
TEMED
10 %
100%
80 µl
110 µl
160 µl
220 µl
•
Load gel solution in a syringe and attach a 0,4µm or 0,25µm sterile filter
•
Pour gel through sterile filter into the glas sandwich.
5.3 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 in a
tight plastic bag.
Note: Prepare electrophoresis unit prior to disassembling the gel cuvette. 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.
14 _______________________________________________________ TGGE MAXI manual
6. Electrophoresis
6.1 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 6.4 and 6.5).
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,
•
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.
Voltage:
100 V - 400 V
start with 300 V
(Current:
5 mA - 25 mA
Approx. 10-20 mA)*
Run Time:
30 min - 4 h
Approx. 3h
*Note 1: The controller is designed to control voltage rather than amperage (set [mA] and
[Vh] to maximum values).
Note 3: To minimize run time in a parallel gel, start with a temperature right below the
melting temperature of the fragment.
6.2 Pre-run for sample loading and temperature equilibration
Let samples migrate into the gel at homogenous temperature. For this purpose a multistep
program can be set up in the controller:
Step 1:
10 minutes, 300Volt, 20°C (L0 and L10)
sample migrate from slots into gel
Step 2:
10 minutes, 0Volt, temperature gradient
temperature gradient equilibrates
Step 3:
main run
samples are separated
For creating a multi step program see section 7.5.
_________________________________ 15
6.3 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 4: Set up of the gel for electrophoresis
6.3.1
Prepare 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:
Ü
parallel or perpendicular gel (see section 5)
Ü
samples in loading buffer (for sample preparation see chapter 9)
Ü
1000 ml running buffer (for recipes see chapter 16.1)
Ü
4 buffer wicks (024-215)
16 _______________________________________________________ TGGE MAXI manual
Ü
1 cover film (024-232)
Ü
thermal coupling solution (0.1% Triton or 0.1% Tween 20, degas carefully)
Ü
gel cover plate
_________________________________ 17
6.3.2
Gel setup for electrophoresis
1)
Adjust electrophoresis chamber with the 4 leveling feet.
2)
Fill 500 ml running buffer in each buffer tank (check orientation of the buffer tanks:
parallel or perpendicular TGGE! see section 6.4 and 6.5).
Note: Wipe off any spilled buffer from the electrophoresis unit. Never run device if buffer
has been spilled.
3)
Soak 4 buffer wicks with running buffer
4)
Disassemble gel sandwich. Clean backside of the gel support film with a soft tissue.
5)
Apply not more than 2 ml of thermal coupling solution (0.1% Triton 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)
Cover the gel with a cover film. The cover film should be placed just beneath the slots.
8)
Attach pre-soaked buffer wicks on top and bottom of the gel (fold 2 sandwiches of
two wicks each in the middle; the folded side should face the gel)
Buffer wick
Gel
Cover film
Gel
Buffer wick
Figure 5: Setup of gel , cover film and buffer wicks.
18 _______________________________________________________ TGGE MAXI manual
9)
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.
10)
Attach gel cover plate (the cover plate should have contact to the wicks, but must not
squeeze gel or wicks).
11)
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.
6.4 Perpendicular TGGE
In perpendicular TGGE one sample is separated over a broad temperature range. This
application is mainly used to check the melting behavior of a sample (see section 10.2). 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 6.
The migration of DNA / RNA molecules is indicated by the arrow on the safety lid of the
electrophoresis unit.
Figure 6: 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.
_________________________________ 19
6.5 Parallel TGGE
In parallel TGGE multiple samples are separated along the temperature gradient. For casting
of the gel use glass plate with 32 slot formers (024-229). The buffer tanks for parallel TGGE
must be positioned as depicted in Figure 7.
Figure 7: 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.
6.6 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 10.2.
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).
20 _______________________________________________________ TGGE MAXI manual
0
1
2
3
4
5
6
7
8
9
1 0
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:
temperature at L6 is 52°C
_________________________________ 21
7. Programming the TGGE controller
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 6.2).
7.1 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
7.2 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
Aé Bê C quit D enter
Scroll through the list with Aé Bê 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 7.9.4.
22 _______________________________________________________ TGGE MAXI manual
7.3 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/,áñ&+.%
Aè BABC C quit D enter
Press B [ABC] to enter the mode for the selection of letters.
name:>
<
ABCDEFGHIJKLMNOPQRST
UVWXYZ –()αδ/,áñ&+.%
Aç Bè C quit D enter
Move to the desired letter with A [ç] and B [è]. Accept highlighted letter with D [enter].
name:>test
<
ABCDEFGHIJKLMNOPQRST
UVWXYZ –()αδ/,áñ&+.%
Aç Bè 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.
7.4 Enter temperatures for the gradient block
1:L0:
A?
L10:
B
C quit D è
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].
_________________________________ 23
1:L0: 30.0
A
B no
L10: 70.0
ok?
C quit D yes
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.
7.5 Enter electrophoresis parameters
1:L0: 30.0
L10: 70.0
time:
El: 0V 500mA 50W
A?
B V*h
C quit D è
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 l minutes l seconds
If you enter a number without “dot” this value will be interpreted as seconds (“300” => 5
minutes). To program minutes enter a “l” after the number of minutes. To enter hours enter l
l after the number. You can also enter any combination of hours, minutes and seconds.
Example: for 1 hour, 30 minutes, 20 seconds enter 1l 30 l 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.
24 _______________________________________________________ TGGE MAXI manual
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:
A?
L10:
B
C quit D è
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 è
7.6 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].
_________________________________ 25
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+
7.7 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+
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.
7.8 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
Aé
Bê
C quit D enter
26 _______________________________________________________ TGGE MAXI manual
You can scroll through the different lines with Aé Bê.
L6 : 54.8
L7 : 58.9
L8 : 62.9
Aé
Bê
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).
7.9 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
3 language
Aé
Bê
C quit D enter
Scroll through the list with Aé Bê.
7.9.1
Print programs
Connect controller to a dot matrix printer. Select option 1 in the above menu and confirm with
D [enter].
7.9.2
Select / de-select signal
Select option 2 in the special functions. Press A [on] to activate the signal, press B [off] to
inactivate signal.
7.9.3
Select language
Select option 3 in the special function screen. Choose between German and English
7.9.4
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
_________________________________ 27
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.
28 _______________________________________________________ TGGE MAXI manual
8. Staining
8.1 Silver staining
Aside from autoradiography silver staining is the most sensitive method for detecting small
amounts of DNA, RNA or proteins in polyacrylamid 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 (e.g. order number 042-400 or 042-500).
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 freshly, 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
_________________________________ 29
Washing
Developing
Rins 30 seconds
then wash 1 min
Rins again 30 seconds
Fresh aqua dest.
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 6.5 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 6.5 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 at room temperature for one day or use a gel dryer at 50°C for at least 3h.
Exposure to an X-ray film.
30 _______________________________________________________ TGGE MAXI manual
8.4
Elution of DNA from the TGGE gel
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.
_________________________________ 31
9. Sample preparation
9.1.1
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.
9.1.2
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.
9.1.3
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.
32 _______________________________________________________ TGGE MAXI manual
10. Optimization of TGGE
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
10.1 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 behavior 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.
10.1.1 Poland analysis
The melting profile of a DNA fragment can be analyzed with a computer program. The
Poland software calculates the melting behavior 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
•
retrieve Tm plot (melting curve)
_________________________________ 33
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.
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: (commaseparated numbers)
34 _______________________________________________________ TGGE MAXI manual
Thermodynamic parameters:
Oligonucleotide
(ß is function of seq.length)
Long double strand
(default: ß=1.0E-3/M)
Dissociation constant ß:
Strand concentration:
(default: 1.0E-6 M)
Temperature step
High temperature
Low temperature limit:
limit: (default: 110.0 size: (default: 2.0
(default: 40.0 °C)
°C)
°C)
Temperature range:
Tm(p=50%) plot 3d plot
3)
Mobility
plot
Melting
curve
Diff. melting
curve
Which graphics do you want:
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.
_________________________________ 35
Figure 8: 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.
10.1.2 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):
cccgc cgcgc cccgc cgccc gcc
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.
10.1.3 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 pairs13,46,47. Crosslinking of the PCR product is done e.g. in a flatbottom 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.
10.1.4 Use of SSCP primers
In many cases primer from SSCP may be used for TGGE analysis. Nevertheless, the resulting
DNA fragments should be checked in the Poland analysis. If there is only one melting domain
add a GC clamp to one of the primers (see section 10.1.2)
10.2 Find correct temperature gradient
Poland analysis gives the first indication, which temperature gradient should be applied for
36 _______________________________________________________ TGGE MAXI manual
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 6.4). 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 9)
Figure 9: 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 6.6.
10.3 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.
electrophoresis
_________________________________ 37
cold
warm
T1
T2
Figure 10: 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 10.5)
10.4 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 11).
38 _______________________________________________________ TGGE MAXI manual
A
3 0 °C
7 0 °C
B
3 0 °C
4 0 °C
Figure 11: 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.)
10.5 What to do, if different samples have very similar melting points:
Heteroduplex analysis with TGGE
10.5.1 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 9.1.3).
_________________________________ 39
a
a
A
a
A
a
a
A
A
a
A
A
denature
re-anneal
Figure 12: 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 12): 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 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 13: 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 10.2). 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
40 _______________________________________________________ TGGE MAXI manual
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 14: 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.
10.5.2 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 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 12. Separation in
parallel TGGE results in up to four different bands (see
Figure 13). 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
_________________________________ 41
11. The TGGE test kit (order number 024-050)
The TGGE test kit 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 typ)
•
the heteroduplex sample (sample has been prepared as described in section 10.5.1)
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 5.2.1).
11.1 Perpendicular TGGE using the Biometra TGGE test kit
1) sample preparation
mix 100 µl heteroduplex sample with
100 µl loading buffer TAE (see section 16.1)
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
6) silver stain gel
42 _______________________________________________________ TGGE MAXI manual
11.2 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 16.1)
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 6.3.2
5) start main run
Temperature gradient
Voltage:
Run time:
4) silver stain gel
30 to 60 °C
400V
3h
_________________________________ 43
12. Technical specification
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
Transformator
450 VA
44 _______________________________________________________ TGGE MAXI manual
13. Ordering information:
TGGE MAXI System:
024-200
Electrophoresis unit with gradient block, controller, power supply, manual,
starter kit
024-204
TGGE MAXI starter kit
1 glass plate perpendicular, 1 glass plate parallel, 2 glass plates without
Spacer, 12 clamps, 2 silicone sealings, polybond film (25), gel cover film (10),
buffer wicks (100), AcrylGlide (100ml)
TGGE test kit
024-050
Test DNA for perpendicular and parallel test runs: wild type DNA (control),
mutant DNA, hetaera duplex DNA, sample buffer.
Self adhesive slot forming units (8 strips with 28 units á 8µl each for parallel
gels, 9 strips with one broad slot á 200µl each for perpendicular gels)
024-121
TGGE MAXI buffer wicks, 100/pkg (18 x 20 cm)
024-215
TGGE MAXI glass plate without Spacer 23.5 x 23.5 cm
024-221
Self adhesive slot forming units for replacement, 28 pcs. á 8µl
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 (1 024-228
slot, 75 µl)
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
_________________________________ 45
14. Trouble-shooting
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
clean Spacer,
Dust on spacers
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 (024-221)
are included in the TGGE maxi system
Acrylamide solution gets behind
the support film during pouring
the gel
Support film is not properly
attached to the glass plate
Fix polybond film with adhesive tape
along the upper edge of the glass plate
Teflon film peels away from the
thermoblock
Block has been cleaned with
strong detergents or aggressive
chemicals
Contact Biometra
No current
Amperage and Wattage have
been set to “0”
Set Amperage and Wattage to maximum
values (Electrophoresis should be
controlled by Voltage)
Current oscillates
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
Gel interpretation
Optimize primer design
No separation of hetero duplex
samples
(parallel TGGE)
Irreproducible gels
Inappropriate fragment
Perform Poland analysis
Wrong temperature gradient
Perform perpendicular TGGE
Acrylamide of poor quality
Use only high quality chemicals (p.a.)
Erratic temperature distribution
over and under the gel
Use only minimum volume of thermal
coupling solution under the gel
46 _______________________________________________________ TGGE MAXI manual
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
_________________________________ 47
15. 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 temperature-gradient 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: 127139
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: 840-847.
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.
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
48 _______________________________________________________ TGGE MAXI manual
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 sequence-specific
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
30.
Hecker R., Wang Z., Steger G. and Riesner D. (1988): Analysis of RNA structure by temperaturegradient 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 non-necrogenic variants by temperaturegradient 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
_________________________________ 49
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
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): Photochem. Photobiol. 40, 29 ff
50 _______________________________________________________ TGGE MAXI manual
16. Appendix
16.1 Buffers
16.1.1 Running buffers:
TBE Running buffer
10 x TBE (stock solution)
TAE Running Buffer
50 x TAE (stock solution)
pH 8.0
MOPS-Running Buffer
50x MOPS (stock
0.1 x conc. TBE (up to 1x conc. TBE is possible)
890 mM Boric Acid
20 mM EDTA
890 mM TRIS
Do not titrate to adjust pH!
1 x conc. TAE, pH 8.0
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
16.1.2 Loading buffers:
Loading buffer TBE
TBE running buffer
0.1% Triton-X 100
0.01% Bromophenol Blue dye
0.01% Xylene Cyanol dye
_________________________________ 51
Loading buffer TAE
TAE running buffer
0.1% Triton-X 100
0.01% Bromophenol Blue dye
0.01% Xylene Cyanol dye
2 mM EDTA
Loading buffer MOPS
MOPS running buffer
1 mM EDTA
0.05% Bromophenol Blue dye
0.05% Xylene Cyanol dye
pH = 8.0
16.1.3 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
52 _______________________________________________________ TGGE MAXI manual
17. Instructions for return shipment
If you would like to send the unit back to us, please read the following return instructions.
Should you have any problems with the TGGE System, please contact your local Biometra
dealer or our service department:
Biometra biomedizinische Analytik GmbH
Service Department
Rudolf-Wissell-Straße 30
D-37079 Göttingen
Phone:++49 – (0)5 51 / 50 68 6-0
Fax: ++49 – (0)5 51 / 50 68 6-66
Return only defective devices. For technical problems which are not definitively recognisable
as device faults please contact the Technical Service Department at Biometra.
Use the original box or a similarly sturdy one.
Label the outside of the box with “CAUTION! SENSITIVE INSTRUMENT!”
Please enclose a precise description of the fault, which also reveals during which procedures
the fault occurred, if possible.
• Important: Clean all parts of the instrument from residues, and of biologically dangerous,
chemical and radioactive contaminants. Please include a written confirmation ( use the
“Equipment Decontamination Declaration” following on the next page) that the device
is free of biologically dangerous and chemical or radioactive contaminants in each
shipment. If the device is contaminated, it is possible that 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 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.
_________________________________ 53
18. Equipment Decontamination Certificate
To enable us to comply with german law (i.e. §28 StrlSchV, §17 GefStoffV and §19 ChemG)
and to avoid exposure to hazardous materials during handling or repair, will you please
complete this form, prior to the equipment leaving your laboratory
COMPANY / INSTITUTE --_______________________________________________________________________
ADDRESS
________________________________________________________________________
TEL NO ________________________
FAX NO _________________________
E-MAIL___________________________________________________________________
EQUIPMENT
If on loan / evaluation
Model
Serial No
______________
__________________
______________
__________________
Start Date: ________ Finish Date ________
Hazardous materials used with this equipment_
__________________________________________________________________________
___________________________________________________________________________
Has the equipment been cleaned and decontaminated? YES / NO (delete)
Method of cleaning / decontamination:
__________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
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.
54 _______________________________________________________ TGGE MAXI manual
19. Warranty
This Biometra instrument has been carefully built, 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 for 12
month as described under the following conditions:
This warranty is valid for 12 month from date of shipment to the customer from Biometra
or an authorized distributor. 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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