3.3.1. Restriction enzyme digestion and agarose gel electrophoresis
This step is carried out in order to array chromosomal sections across a one-dimensional space so that unique sections can be probed for matches to a query sequence. In principle, the targeted gene will be embedded in a single chromosomal segment flanked by specific sequences that match the restriction enzyme used.
- Digest 5-10 mg of genomic DNA in a
volume of 30 ml with an appropriate restriction enzyme by
setting up reaction as follows:
3 ml 10X buffer,
0.3 ml of BSA if needed (this will be on the restriction enzyme label),
3 ml enzyme (10U/ml),
5-10 mg genomic DNA,
Add sterile water to reach a total volume of 30 µl.
Generally, enzymes that cut frequently in the target genome are used here (e.g., ‘four-cutters’ that cut at a specific four-base-pair sequence, an event expected to occur ca. once every several hundred base-pairs).
- Allow the digestive reaction to go for overnight at 37°C (or temperature appropriate to your specific enzyme).
- Run the full 30 µl of reaction mixture with 3 µl 6X loading dye on 1% agarose gel (see section 3.2.1) containing ethidium bromide (1µg/ml ) for 2 hours at 100 volts. Include one lane of a DIG-labelled DNA Molecular Weight Marker at the appropriate level.
- Take a picture of the digestion.
- Depurinate the agarose gel for exactly 10 min in 0.25 M HCl if DNA fragment > 4 kb.
- Denature the gel in freshly made denaturing
solution (0.5M NaOH, 1.5 M NaCl) for 2 x 15 min at RT, slowly shaking
on rotating shaker.
Denaturation of the DNA into single strands allows hybridization with a probe possible.
- Rinse the gel with sterile water.
- Neutralize the gel in neutralizing solution (0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl) for 2 x 15 min, slowly shaking on rotating shaker.
- Equilibrate gel in 20X SSC for 10 min.