Agagel maxi 020

Agagel maxi 020
Agagel Maxi
Code-No. 020-300
Manual
Juli 2003
!! Warning !!
Please read these instructions carefully
before using this apparatus!
Biometra GmbH
Rudolf-Wissell-Str. 30
D-37079 Göttingen
Postfach 1544
D-37005 Göttingen
Tel: ++49 – (0)5 51 / 50 68 6-0
Fax: ++49 – (0)5 51 / 50 68 6-66
e-mail: [email protected]
internet: http://www.biometra.de
Service Department
Rudolf-Wissell-Str. 14-16
D-37079 Göttingen
Tel.: ++49 – (0)5 51 / 50 686-10
Fax.: ++49 – (0)5 51 / 50 881-11
[email protected]
Index
1
2
3
4
5
6
7
8
page
Intended uses and specifications ...................................................................................... 3
Set up .................................................................................................................................. 4
2.1 Unpacking and Checking.............................................................................................. 4
2.2 Electricity Supply.......................................................................................................... 4
2.3 Location ........................................................................................................................ 4
2.4 Chamber Adjustments................................................................................................... 4
2.5 Comb Selection and Adjustments................................................................................. 4
2.6 Buffer Preparation......................................................................................................... 5
2.7 Gel Preparation ............................................................................................................. 5
2.8 Gel Casting and Chamber Preparation.......................................................................... 6
Operation............................................................................................................................ 6
3.1 Checking the System before Loading (optional) .......................................................... 6
3.2 Sample Preparation and Loading .................................................................................. 6
3.3 Electrophoresis.............................................................................................................. 7
Photography of the Gel ..................................................................................................... 7
Cleaning.............................................................................................................................. 8
Electrophoresis buffers and solutions.............................................................................. 8
Troubleshooting ................................................................................................................. 9
Service............................................................................................................................... 10
IMPORTANT !
Please read these instructions carefully before using this apparatus!
2
1 Intended uses and specifications
The Agagel Maxi is intended to be used for the electrophoretic fractionation of DNA and
RNA molecules in an agarose gel matrix. The system comes complete with all the accessories
needed to cast and run 18 cm long, 20 cm wide and 1 cm thick gels. The accessories include
one UV transparent gel tray (20 cm long, 20 cm wide, 1.5 cm deep) with a ruler, one UV
transparent gel tray (10 cm long, 20 cm wide, 1.5 cm deep), one set of rubber casting dams,
two combholders, two 40/22 well combs, two 10/2 well combs, one depth/leveling gauge, one
main chamber with built-in bull’s-eye level and leveling feet, one interlocking safety cover
with leads, and an instruction manual.
3
2 Set up
2.1
Unpacking and Checking
Unpack and carefully examine the chamber and the accessories; the interlocking safety lid can
be removed for examination by sliding it off (see Figures 1). Report any damage to Biometra,
and save all packing material if damage is found. Do not attempt to use this apparatus if it is
damaged.
2.2
Electricity Supply
This apparatus has been designed to be connected with an electrophoresis power supply. The
apparatus itself must not be connected to earth ground.
Do not exceed 300 volts or 30 watts of applied power
(watts = volts x amps [1amp = 1000mA]).
Do not use the apparatus if any of the following conditions exist:
— Buffer leaks from the main chamber.
— Cracks are present in the chamber or safety cover.
— Worn or frayed electrical connecting cables are encountered.
2.3
Location
Place the apparatus in proximity to the electrophoresis power supply with which it is to be
connected. Make sure that this is a safe and dry location at a safe distance from the edge of
the working surface.
2.4
Chamber Adjustments
Use the adjustable leveling feet to center the bubble in the built-in bull’s-eye level; this
process is most easily accomplished by first screwing the leveling feet all the way in and then
checking that the working surface is level. Adjustments may need to be repeated if the
chamber’s position is changed. Place both rubber casting dams on the ends of the gel tray and
place the tray in the chamber.
Note: There is an interlock that keeps the tray in place.
2.5
Comb Selection and Adjustments
Select the comb with the appropriate number of wells for your experiment and attach it to the
combholder using the depth adjustment screws, usually the combs with 40 or 22 wells are
used when multiple samples are analyzed. Place the combholder with the comb onto the tray
so that each end of the combholder rests in a groove on one of the tray’s edges; the
combholder should be resting parallel to the rubber casting dam near the ruler at the cathodal
end of the tray. Use the depth leveling gauge to maintain an even spacing between the bottom
of the comb’s teeth and the surface of the gel tray while the depth adjustment screws are being
tightened. The 0.75mm thin spacer on the leveling gauge is used in most cases, but when very
fragile gels are cast, the 1.5mm spacer is recommended.
4
2.6
Buffer Preparation
Prepare 1800ml of electrophoresis buffer. Buffer recipes are described in Section 6.
2.7
Gel Preparation
Select from Table 1 the optimal concentration of agarose required for the separation of DNA
molecules in the size range being analyzed. Various types of agarose are currently in use, and
it is important to evaluate the suitability of the type being used before valuable samples are
committed to a gel.
Table 1
Agarose concentrations recommended for the separation of DNA fragments of various sizes.
Agarose
concentration
0.5%
0.7%
1.0%
1.2%
1.5%
2.0%
Size of DNA fragments (kb)
1.0
0.8
0.5
0.4
0.2
0.01
to
to
to
to
to
to
30
12
10
3
3
1
Table 2 summarizes the buffer volumes needed to cast gels of various thicknesses (1.0, 0.5
and 0.25cm). The buffer volumes needed to fill the chamber to the appropriate level are also
indicated. Weigh out the appropriate amount of agarose for the selected concentration and
volume, and pour it into an Erlenmeyer flask containing the predeter-mined volume of
electrophoresis buffer. The agarose is heated and stirred on a hot plate, or heated in a
microwave oven, until it boils and is completely dissolved. Distilled water may have to be
added to replenish what has boiled off; the amount of water that has boiled off can be
quantitated by weighing the flask including the agarose and buffer before and after the boiling
step. Discontinue heating the agarose and allow it to cool to approximately 80 °C. Unlike
other trays on the market, with the new tray the agarose does not have to be cooled to 65 °C.
This lets you pour gels faster than ever. The new tray is heat resistant and will easily
accommodate agarose at 80 °C.
Table 2
Buffer volumes required to cover gels of various thicknesses.
Gel thickness
1.0cm
Volume of agarose
in the gel tray
300ml
Buffer volume
in the chamber
1400ml
0.5cm
170ml
1290ml
0.25cm
85ml
1240ml
5
2.8
Gel Casting and Chamber Preparation
Pour the entire predetermined volume of molten agarose solution into the gel tray. Carefully
remove any trapped air bubbles from the gel solution by using a glass or plastic probe. Allow
the gel to cool and solidify completely. The comb can be removed more easily from the gel if
a few ml of water or electrophoresis buffer are pipetted onto the juncture between the comb
and the gel. Carefully remove the comb by gently pulling the combholder straight up while
holding it at both ends. Carefully remove both rubber end blocks and return the gel tray to the
chamber so that the sample wells are closer to the cathode (negative electrode, black lead)
than to the anode (positive electrode, red lead). Slowly fill the chamber with electrophoresis
buffer until the upper surface of the gel is submerged about 1mm; refer to Table 2 for the
buffer volumes required for gels of different thicknesses.
To recirculate the electrophoresis buffer, the two quick-disconnect connectors can be inserted
into the recirculation ports in the anodal and cathodal buffer compartments. Hoses leading to
and from a pump can then be easily attached.
3 Operation
3.1
Checking the System before Loading (optional)
Carefully slide the interlocking safety cover onto the chamber and make sure that both banana
plugs are securely attached. Connect the attached leads to the electrophoresis power supply
making sure that the polarity of the leads agrees with the polarity of the output. Refer to Table
3 to select an appropriate voltage, switch the power supply on and adjust the output to the
desired level. Check that the system is functioning properly before you commit valuable
samples to the gel. The voltage and current levels should correspond to the expected levels
and should not fluctuate, and bubbles should rise from the electrodes in the chamber. After
verifying that the system is operating properly, switch the power off and disconnect the leads
from the power supply.
Table 3
Minutes required by markers to move through 6.2cm of a 1%, 0.5cm thick agarose gel.
1 X TBE (with EtBr)
Marker
fragment
size (bp)
50
100
500
1000
50V
48mA
115
120
165
218
100V
56
60
70
88
1 X TAE (with EtBr)
150V
121mA
32
34
50
60
50V
80mA
102
102
121
145
100V
49
49
60
72
150V
219mA
30
30
36
42
3.2
Sample Preparation and Loading
Table 4 lists the maximum sample volumes that can be applied to the wells formed by 1mm,
1.5mm and 2mm combs without overfilling the wells and spilling the samples. Taking this
information into consideration, use an air displacement pipet and disposable pipet tips to
combine appropriate volumes of loading dye and DNA or RNA solutions to prepare your
samples. Samples can be conveniently prepared in microcentrifuge tubes, Microtiter™ trays
or on Parafilm. Remove the interlocking safety cover from the chamber and carefully load the
6
samples into the individual wells using the same pipetting device. Make sure that the gel and
gel tray are aligned properly so that the ends of the gel are parallel to the platinum electrodes,
and then replace the interlocking safety cover.
Table 4
Sample volumes recommended for combs with wells of various sizes.
Please be aware that the maximum possible volume will vary in dependence from gel
concentration!
comb type
comb
thickness
2 of 2/10
1.0mm
10 of 2/10
1.0mm
22 of 22/40
40 of 22/40
2 of 2/10
1.0mm
1.0mm
1.5mm
10 of 2/10
1.5mm
22 of 22/40
40 of 22/40
22 of 22/40
40 of 22/40
1.5mm
1.5mm
2.0mm
2.0mm
tooth type Sample volumes for different gel
thicknesses
1.0cm
0.5cm
0.25cm
large well
600µl
300µl
150µl
reference
50µl
20µl
10µl
large well
150µl
60µl
30µl
reference
30µl
13µl
5µl
small well
51µl
26µl
13µl
small well
21µl
10µl
4µl
large well
800µl
400µl
100µl
reference
35µl
18µl
7µl
large well
150µl
80µl
25µl
reference
35µl
18µl
7µl
small well
80µl
44µl
15µl
small well
35µl
19µl
6µl
small well
100µl
60µl
25µl
small well
55µl
27µl
11µl
3.3
Electrophoresis
Connect the leads to the electrophoresis power supply, switch the power on and adjust the
output to the desired level as outlined in Section 3.1 and Table 3. Check that the system is
functioning properly and proceed with the electrophoretic fractionation for the required period
of time. The migration of the dye front is used to monitor the extent of the molecules’
migration. Switch the power supply off at the end of the run, and disconnect the leads from
the power supply before sliding off the interlocking safety cover.
4 Photography of the Gel
Remove the gel tray with the gel from the chamber and take care that the gel does not slide off
the tray. Stain the gel with ethidium bromide (EtBr) and destain it, if necessary. Photographs
of the fluorescent ethidium bromide–stained DNA or RNA molecules in the gel can be taken
through the UV transparent gel tray when it is illuminated by a UV transilluminator. The rule
on the underside of the gel tray facilitates subsequent mobility measurements on the
photographs.
7
5 Cleaning
The tray is extremely chemical resistant. This makes it ideal for RNA work where the
removal of RNases is critical. To clean the tray, wash the entire tray with a mild laboratory
detergent. For critical work, the inside of the tray should be wiped out with a lint-free tissue
soaked in ethanol or acetone, and then rinsed in DEPC water or allowed to air dry.
Do not use alcohol or organic solvents to clean the chamber, the lid or any of the
accessories. Use only mild non-abrasive detergents.
Remove RNases by soaking the chamber and the tray in a 0.1% solution of diethyl
pyrocarbonate (DEPC) for 2 hours at 37 °C. Do not soak the combs, depth leveling gauge or
combholder in depc containing solutions. To remove RNase from these items soak in a 1.0M
NaOH solution for 30 minutes and rinse with DEPC–treated water that has been previously
autoclaved. Air dry the chamber and accessories at room temperature or 37 °C. An alternative
protocol would involve soaking the chamber, gel tray and accessories in 1.0M NaOH for 30
minutes, followed by rinsing in DEPC–treated water that has been previously autoclaved. The
gel tray may be cleaned with ethanol.
6 Electrophoresis buffers and solutions
5X TBE buffer
Stock
(2 liters)
(0.45M Tris, 0.45M Boric Acid, 10mM EDTA)
Tris base, mw 121.1
109.0g
Boric acid, mw 61.8
55.6g
EDTA (free acid) mw 292.2
5.8g
Deionized or distilled water to
2000ml
Make sure that you use Tris base and the acid form of EDTA. It is not necessary to adjust the
pH of this buffer; the pH should be about 8.3.
1X TBE buffer
Working
Solution
Tris borate
EDTA
90mM
2mM
50X TAE buffer
Stock
(1 liter)
(2.0M Tris, 1M Acetate, 100mM EDTA)
Tris base, mw 121.1
242.0g
Glacial acetic acid, mw 17.4M
57.1ml
EDTA (disodium, dihydrate), mw 372.2
37.2g
Combine the Tris base and the EDTA first, add deionized or distilled water to about 850ml,
dissolve the reagents and then add the acetic acid. Check and adjust the pH to 8.5 if necessary
and add deionized or distilled water to 1000ml.
1X TAE buffer
Working
Solution
Tris
Acetate
EDTA
40mM
20mM
2mM
6X Loading
Dye Mix
Bromophenol blue
Xylene cyanol
Sucrose in 50mM EDTA (pH 8)
Store this solution at 4 °C.
8
0.25%
0.25%
40%
Ethidium bromide stock (10 milligram per ml)
Ethidium bromide
Sterile deionized water
Store this solution in the dark.
100
10
mg
ml
Ethidium bromide working solution (0.5 microgram per ml)
Stock solution (10mg/ml)
50µl
Electrophoresis buffer
1000ml
If a gel is to be stained with ethidium bromide after electrophoresis, the same 0.5 microgram
per ml solution can be used. Staining can take up to one hour if thick gels are used. Destaining
in water or in 1mM magnesium sulfate takes about 30 minutes. Ethidium bromide is a
powerful mutagen. Wear gloves while you work with the solutions, buffers and gels
containing ethidium bromide.
7 Troubleshooting
Problem: The wells collapse.
Explanation: Wells may collapse when the comb is removed either because the agarose
adheres to the comb, or because the agarose is not strong enough to maintain the well when
low percentage agarose gels or low melting temperature agarose gels are cast.
Solution: To minimize adhesion of the agarose to the comb, it is advisable to remove the
comb as soon as possible after the agarose has cooled and solidified completely. If the agarose
is allowed to dry on the surface it will tend to stick to the comb. The addition of water or
electrophoresis buffer to the juncture between the comb and the agarose will prevent the gel
from drying. When the comb is removed, water and not air bubbles will seep into the well and
support the walls of the well. When low melting agarose gels are cast, it may be necessary to
work in a cold room so that the comb’s removal does not destroy the wells.
Problem: The gel sticks to the rubber casting dams and causes the gel to tear where the
wells have been formed.
Explanation: If the gel has dried it also tends to stick to the rubber casting dams.
Solution: A spatula can be run along the juncture between the gel and the rubber casting
dams before these are removed from the gel tray.
Problem: The leads are connected to the electrophoresis power supply but no current
flows through the system.
Explanation: Usually a broken electrode wire or a faulty connection can account for this
problem.
Solution: Check the leads, connections and platinum electrodes. Contact Biometra if a broken
electrode or lead is encountered; the electrodes and leads can be replaced.
REFERENCES
Ausubel, S.M., Brent, R., Kingston, R.R., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. 1987. Current
Protocols in Molecular Biology. John Wiley
& Sons, New York, NY.
Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd Edition. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
9
8 Service
Should you have any problem with the device, please contact our service division or
your local Biometra dealer:
Biometra
GmbH
Service Division
Rudolf-Wissell-Straße 14-16, D-37079 Göttingen
Phone: ++49 – (0)5 51 / 50 88 1-10 or -12
Fax:
++49 – (0)5 51 / 50 88 1-11
E-mail: [email protected]
Please follow the return instructions when returning the device:
Return instructions
♦ Return only faulty devices. For any technical problem that is not clearly identifiable as a device
failure, please contact the technical support of Biometra (Phone: ++49 (0) 551/50 88 1 - 10 or 12).
♦ Use the original packing or a similar robust packing when returning the material.
♦ Mark the outer packing with “CAUTION! SENSITIVE ELECTRONIC INSTRUMENT!”
♦ Please include a precise description of the problem and preferably one that also points out by
which procedure the failure occurs or where it originates.
♦ Important:
Clean every part of the device from residues such as chemical, radioactive and dangerous
biological contamination. Please confirm in writing (Equipment Decontamination
Certificate) before every return that the device is free of radioactive and dangerous biological
contamination. Contaminated devices sent to Biometra will be refused!
♦ The sender will be liable for the repair order in case of damage resulting from an inadequate
decontamination of the device.
♦ Please include a note with the following information:
a) Sender’s name and address
b) Contact person and phone number in case of inquiry.
10
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, 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.
11
Warranty
This Biometra device has been accurately produced and the quality thoroughly controlled before
delivery and is therefore guaranteed to correspond to the product specifications in this manual.
Biometra offers a 12-month warranty on defective parts under the following conditions:
This warranty is limited to the product and, if applicable, to the standard original
accessories. It is presumed that this device will exclusively be operated in accordance
with Biometra’s instructions.
Defective parts returned within the warranty period will be repaired or replaced without any charge by
Biometra. This warranty does not cover damage caused by misuse, neglect or normal wear.
______________________________________________
Biometra GmbH
Rudolf-Wissell-Straße 30, D-37079 Göttingen
P.O. Box 1544, D-37005 Göttingen
Phone: ++ 49 (0)5 51 / 50 68 6-0
Fax: ++ 49 (0)5 51 / 50 68 6-66
E-mail: [email protected]
Internet: http://www.biometra.de or http://www.biometra.com
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