Agagel Standard 020

Agagel Standard 020
Agagel Standard with cooling
Code-No. 020-100
Agagel Standard without cooling
Code-No. 020-200
Manual
April 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 88 1-10
Fax.: ++49 – (0)5 51 / 50 88 1-11
[email protected]
1.0 INTENDED USES AND SPECIFICATIONS
The Biometra Agagel Standard with cooling and Agagel Standard without cooling are
intended to be used for the fractionation of DNA and RNA molecules in an agarose gel matrix. The
system comes complete with all the accessories needed to cast and run 10 x 10 cm gels.
Accessories included with the system are:
- one main chamber,
- one interlocking safety cover with leads,
- one 10 x 10 cm UV transparent gel tray with 2 casting gates,
- two 18 well combs (1mm),
- one manual and
- one Warranty Certificate
Accessories:
Combs:
Comb,
Comb,
Comb,
Comb,
3 wells,
12 wells
12 wells
18 wells
170 µl per lane*
35 µl per lane*
80 µl per lane*
32 µl per lane*
1 mm thick,
1 mm thick,
2 mm thick
1 mm thick
Code-No. 020-201
Code-No. 020-202
Code-No. 020-203
Code-No. 020-207
(* = Max. volumes recommended for 10 mm thick gels)
Gel trays:
Agagel Standard UV-transparent gel tray,
Agagel Standard UV-transparent gel tray
with 2 casting gates
Casting gates for Code-No. 020-205, 2/pkg
1
10 x 10 cm
Code-No. 020-204
10 x 10 cm
Code-No. 020-205
Code-No. 020-206
2.0 SAFETY
Attention:
Do not use organic solvents (e.g. alcohols, acetone) to clean
the gel apparatus.
Do not heat the apparatus over 50°C.
Allow agarose to cool down to approximately 65°C before
pouring the gel.
Danger! High voltage!
3.0 SET UP
3.1 Unpacking and Checking
Unpack and carefully examine the electrophoresis chamber and accessories. Report any damage to
Biometra. Do not attempt to operate this device if it is damaged. Save all packing material if
damage is found.
!! Attention !!
Please fill out and send back the warranty registration card. This is
important for you to claim to full warranty: both parts and repair are
covered within the full warranty period!
3.2 Electricity Supply:
This electrophoresis chamber has been designed to operate with D.C. current.
Do not exceed 15 watts.
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WARNING: THIS EQUIPMENT MUST NOT BE EARTHED
3.3 Important Electrical Safety Notes:
Do not operate this equipment if any of the following conditions exist:
- If buffer leaks from the main electrophoresis chamber.
- If cracks are present in the electrophoresis chamber or safety cover.
- If the electrical connection cables are warm or frayed.
4.0 LOCATION
Place the chamber in proximily to the Power Pack with which it is to be connected. Be sure to place
the chamber in a safe, dry location away from the edge of the working surface.
5.0 HANDLING
5.1 Chamber Adjustment
Level the chamber at the location where the gel will run. Seal the gel tray with an adhesive tape or
hot agarose (max. 50ºC) at both sides and place the tray in the chamber.
5.2 Buffer Preparation
Prepare the running buffer (see Section 6.0). For staining the DNA ehtidium bromide can be added
to the running buffer, it intercalates during the run into the DNA. The separation can be observed
during the run by placing the gel tray on a transilluminator. But the electrophoretic mobility of the
DNA is reduced by about 15%.
5.3 Gel Preparation
Select from Table I the appropriate concentration of Agarose required to optimize separation:
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TABLE I:
Agarose %
Size of Fragments to be Separated (kb)
---------------------------------------------------------------------------------------------------------------------0.5
1 to 30
0.7
0.8 to 12
1.0
0.5 to 10
1.2
0.4 to 3
1.5
0.2 to 3
2% (Sieving Agarose)
0.01 to 1
For casting a 4 mm thick gel, combine the buffer solution with Agarose (we recommend Amresco
Agaroses) in the desired concentration to form a total volume of 50 ml. Heat this while stirring in a
hot water bath (or use a microwave oven) until it boils and the agarose is completely dissolved.
Discontinue heating and allow liquid to cool to approximately 65ºC to 50ºC before pouring.
5.4 Gel Casting and Chamber Preparation:
Pour the still molten Agarose solution into the gel tray filling up to approx. 4 mm height.
Position one or two combs into the slots of the gel tray. Caution should be used to ensure the
position of the comb is at 90º with respect to either side of the gel tray. This will ensure that each
sample well will be the same distance from the end of the gel.
Allow the gel to completely cool and solidify (30 - 45 min.). Carefully remove the comb(s) by gently
pulling it straight up while holding on the top of the comb from both sides. Carefully remove the tape
(or Agarose) from both sides of the gel tray.
Slowly fill the chamber with buffer solution until the top of the gel is submerged approximately 1 mm.
5.5 Sample Preparation and Loading:
Combine the DNA or RNA sample and loading buffer (see Section 4), e.g. 5 parts sample solution
(100 - 500 ng DNA or approximately more of ss DNA because of the worse staining) to 1 part 6x
loading buffer, end volume 15 µl.
Once combined, carefully load sample into each well (use a syringe). Following this, gently pull the
gel tray up against one side of the chamber thus ensuring that both end of the gel are parallel to the
platinum electrodes.
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5.6 Connect Chamber to Power Pack:
Carefully place the safety cover onto the chamber ensuring that both safety plugs are securely
attached. Connect the attached leads to an appropriate electrophoresis Power Pack (Biometra
Power Pack P20 or P25). Turn on the Power Pack and adjust the desired level: normally 100 - 150
V, higher voltages require water cooling (
max. water pressure = 0.5 bar).
DNA is negatively charged and runs to the red marked anode. Run electrophoresis until the
bromophenol blue band has reached the end of the gel.
5.7 Disconnection From Power Pack
Before removing the safety cover from the chamber, first turn off the Power Pack and disconnect the
leads from the Power Pack.
5.8 Gel Photography
Photographs of ethidium bromide stained gels may be taken through the gel tray when placed on a
UV transilluminator. The gel tray is only 95% transparent so adjust to the normal exposure settings
may be necessary.
If the gel has a high background destain it with 1 mM MgSO4 for 1 hour.
5.9 Cleaning
Use only mild non-abrasive detergents. DO NOT use alcohol or organic
solvents on this product.
For RNase decontamination soak the chamber, gel tray and accessories in a 0.1% solution
of diethyl pyrocarbamate (DEPC) for 12 hours at 37ºC, rinse thoroughly with sterile RNase
free distilled water and dry. Be sure that DEPC is diluted completely before
using the solution.
Any alternate protocol would involve soaking the chamber and combs in 10 M NaOH for
30 minutes, followed by rinsing in DEPC treated RNase free water.
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6.0 SOLUTIONS:
5X TBE BUFFER: (0.45 M Tris, 0.45 M Boric Acid, 10 mM EDTA)
Tris (Base)
109 g
Boric Acid
55.6 g
EDTA (Free Acid)
5.8 g
bidistilled Water
fill up to 2000 ml
Be certain to use Tris base and the acid form of EDTA.
It is not necessarry to adjust the pH of this buffer
1X TBE WORKING SOLUTION (Running BufferJ
Tris
90 mM
Boric Acid
90 mM
EDTA
2 mM
50X TAE BUFFER:
Tris (Base)
242 g
Glacial Acetic Acid
57.1 ml
EDTA, Disodium Salt, Dihydrate
37.2 g
pH
8.5
bidistilled Water
fill up to 1000 ml
1X TAE WORKING SOLUTION (Running Buffer):
Tris
40 mM
Acetate
40 mM
EDTA
2 mM
6X GLYCEROL GEL LOADING BUFFER
Glycerol
40%
Bromophenol Blue (1% in water)
0.25%
Xylene Cyanol (1% in water) (optional)
0.25 %
Store at 4°C
6X SUCROSE GEL LOADING DYE
Sucrose 50 mM EDTA
Bromophenol Blue (1% in water)
Xylene Cyanol (1% in water) (optional)
Store at 4°C
40%
0.25%
0.25 %
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STOCK SOLUTION ETHIDIUM BROMIDE (1 mg/ml)
Ethidium Bromide
100 mg
bidistilled Water
fill up to 2000 ml
Store in a dark bottle at 4ºC.
ATTENTION: Ethidium bromide is suspected carcinogen! Handle it with extreme
care and always wear gloves! Protect your eyes with special glases! Dispose of used
solutions and gels according the instructions appropriately!
1% agarose gel in 1X Working Solution (1 gel)
agarose
0.5 g
1X Working Solution
50 ml
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7.0 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.
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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.
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8.0 NOTES
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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 valid for 24 months from date of shipment to the purchaser through Biometra or an
authorized dealer and can not be assigned to a third party without Biometra’s written agreement.
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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