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Elaine Pinheiro and Aurora Burds Connor, Jan 2006
This protocol works very well with Hybond-XL membrane (Cat # RPN303S for a roll) from GE Healthcare (formerly Amersham).
Note: Genomic DNA should not be stored frozen. Always store at 4°C. It may be very globby, so you can put it at 55°C for an hour or 2 before prepping your digestion and this will make it more manageable.
1. Digest your genomic DNA, using about 10ug per reaction in 100uL total digestion volume. Digest overnight, spiking with a bit more enzyme for 2 more hours in the morning. Concentrate your sample by precipitation: adding 10uL of 3M NaOAc and then 110uL isopropanol, invert tube and spin 10min. Remove liquid carefully, let dry 10 min and reconstitute in 30uL TE. Also digest 1ng of your BAC, if you have one, as a positive control. If you use too much, it will bind all your probe and overwhelm your blot, so 1ng (yes, nano-gram) is plenty.
Some people prefer to concentrate the DNA after completed digestion by simply placing the tube in a speed-vac. This does also concentrate all the salts (which the EtOH wash removes) but they do not seem to harm anything.
2. Separate the DNA sample on low percentage gel (0.6% to 0.8%) agarose gel in 1X TAE. TAE is better for resolving larger fragments. Add ethidium to buffer near red (+). (0.1ug/ml ethidium should be included in the gel for visualization).
3. Run gel 5-6 hours at 40 to 60 volts. Once gel is finished running, be sure to take a picture with ruler. (Line-up ruler with wells). Each well should show a long, even smear of DNA. (BAC lane will not show a smear because there is so little DNA)
Mark membrane with pencil or VWR marker so that you can figure out orientation later. Some people like to trim off the top left corner of the gel and membrane, indicating where the marker lane will be. May sure there is plenty of 20X SSC in tray. Let go for at least 4-6 hours or overnight. Might need to change paper towels after a couple of hours.
9. Rapid Hybridization. Rinse blot briefly in 2X SSC. Crosslink (Stratagene UV Crosslinker - Blot is stable at room temp.) Heat Pre-Hyb Solution to 55-65C. (ExpressHyb works well as the Pre-Hyb and Hyb- from BD Biosciences/Clontech Cat#8015-2) Add 20ml of Pre-Hyb Buffer to 30 cm tube. Pre-hyb for at least 1 hour in 65C oven.
10. Hybridization. Add 1X106 cmp/ml of denatured probe (100C for 5 min.) to 5-10ml of pre-heated Hyb Buffer (5X 106 to 1X 107 cpm total) Discard "old" prehyb buffer and incubate at 65C for at least 6 hs. (ON okay).