Genomic DNA Digest
- 10 µl DNA (10 µg)
- 3 µl 10X Buffer
- 1-2 µl Restriction Enzyme (40 units)
- 3 µl 10mM Spermidine-HCl
- 3 µl 1 mg/ml BSA
- 10 µl H2O
- Total volume: 30 µl
Note: Use DNA from a standard tail prep resuspended in 100 µl. You may want to spec the DNA pf 2-3 samples to determine an average volume for 10 µg DNA rather than just using 10 µl.
- Incubate O/N at 37°C in 96-well plate (or small PCR tubes). Seal edges of plate with Parafilm®. Place damp paper towel on top of plate (or tubes) and wrap the whole thing in plastic wrap. Place in 37°C chamber. (This should prevent condensation in the caps of the tubes.)
- Run entire reaction mix on 0.8% TAE gel. (Medium-sized gel: 150V ~2.5hrs)
- Take a picture of the gel with a ruler along the side. Place the 0-cm mark at the bottom of the wells so you can determine where molecular weight markers are via ruler.
- Soak gel in 0.1N HCl (stock = 14.4N) for at least 20 minutes on shaker.
- Soak gel in 0.4M NaOH for 10-20 minutes on shaker.
- Set Up Capillary Transfer
Precut GeneScreen® membrane and 4 pieces of Whatman® paper.
Fill container with 0.4M NaOH, place glass plate across it and drape a long piece of Whatman across it so its ends are in the NaOH in the container.
Set up transfer, beginning at the bottom, as follows:
Top of transfer
Wrap all edges with plastic wrap to prevent bypass of the capillary,
and stack paper towels on top to pull the NaOH through the gel.
Place book or weight on top.
2 pieces Whatman paper
Membrane (mark wells with 25g needle)
Gel, bottom side up
2 pieces Whatman paper, cut to size of gel, prewet in NaOH
Bottom of transfer
Leave at RT O/N.
- Next day, remove paper towel and top 2 pieces of Whatman paper. Make sure wells are marked with needle and clip edge of what is the front top left corner for orientation.
- Wash membrane in 0.2M Tris, pH 7-8 / 2X SSC for 5 minutes. Then crosslink membrane with UV crosslinker.
- Blot off extra liquid, then roll membrane and place in hybridization tube with DNA side inward. Prehybridize with 15 ml Rapid Hyb™ 15-30 minutes at 65°C.
Note: Make probe either that morning or the day before.
- Pour off prehyb solution and add 20 ml fresh Rapid Hyb™ Buffer with 2-3 X 105 cpm/ml probe.
- Boil probe for 5 minutes, then cool on ice for 5 minutes before adding to hyb solution.
- Hybridize 1.5 - 3 hours at 65°C (You can let it continue O/N).
- Discard hyb and rinse tube with 10 ml 2X SSC, and discard. Then wash in 25 ml 2X SSC for 10 minutes at RT.
- Wash 2X with 25 ml 15 minutes each at 65°C 1X SSC/0.1% SDS, and discard.
- Wash 2X with 25 ml 15 minutes 65°C 0.5X SSC/0.1% SDS, and discard.
- Wash 2X with 25 ml SSC 10 minutes at RT.
- Blot off extra liquid, wrap in plastic wrap and put in phosphoimaging cassette (1 hour) or film (3 days to 1 week).
Pour boiling 0.1% SDS onto membrane, then let cool to RT on benchtop.
Let cool to room temperature while shaking.
Check on phosphoimager.