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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES
REFERENCE NO: AHC/1998/3/3.1/2.3 Appendix 2
TITLE: RESTRICTION ENZYME DIGEST OF GENOMIC DNA
Restriction enzyme digests, usually Hinf1, are carried out on samples for DNA fingerprinting. All DNA should be dissolved and quantified prior to setting up digests.
- a For Hinf1 digests 10mg of genomic DNA is digested with 80 units of enzyme (10U/ml) and 4ml of 10x enzyme buffer. Digests are made up to 42ml with sterile distilled or deionised water.
Xml (10mg) DNA + 8ml enzyme + 4ml 10x enzyme buffer + (30-x)ml water = 42ml
AHC/1998/3/3.1/2.3 Appendix 3)
Add 2ml of gel loading mix A (protocol AHC/1998/3/3.1/2.3 Appendix 4) to the remaining diluted digested DNA and electrophorese for 3 to 4 hours at 75V in parallel with standard markers (i.e. Hind III digest of l phage DNA)
Photograph gel on a UV - transilluminator.
Record any errors such as loss of DNA from wells during loading.
Make a note of incomplete digests. These should be re-digested following re-extraction and precipitation
Successfully digested DNA may now be stored at <-10°C or used on the same day (protocol AHC/1998/3/3.1/2.3 Appendix 9).
- b For enzymes other than Hinf1 10mg of genomic DNA is digested using 50 units of enzyme + 4ml of 10x enzyme buffer. Digests are again made up to 42ml with sterile distilled water.
- Record all details including DNA number, all batch numbers, operator and date.
- Mix and briefly microfuge digests (5 to 10 sec, at maximum speed)
- Incubate for 30 to 60 min at the appropriate temperature. Remix and briefly spin digests (as above) and incubate overnight at the appropriate temperature. For Hinf1 the incubation temperature is 36 to 38°C.
NB: Some enzymes require different incubation temperatures and times.
- Following incubation, mix and centrifuge digests as in 3.
- Quantify the concentration of digested DNA
- Prepare a minigel (0.8% agarose, protocol
Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
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