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.
Procedure
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)
4. Process the gel for blotting. Solutions below are as described in the Hybond-HL instruction manual. Recipes for each are listed below. Between each step rinse the gel in distilled water.
5. Depurination. Place the gel in 0.125M HCl so that the gel is completely covered (submerged) in the solution. Agitate gently for approximately 10 minutes.
6. Denaturation. Submerge the gel in sufficient denaturation buffer. Incubate for 30 minutes with gentle agitation.
7. Neutralization. Submerge the gel in sufficient neutralization buffer. Incubate for 30 minutes with gentle agitation.
8. Transfer. In a big tray set up the following (in layers, starting from the bottom):
- Big sponge soaked in 20X SSC
- Two sheets of whatman filter paper cut slightly bigger than gel size
- Gel
- Saran wrap or parafilm to cover edges of gel and prevent wicking
- Membrane (Hybond XL) cut to gel size
- Two sheets of whatman filter paper cut slightly bigger than gel size
- Big stack of paper towels (6+ inches)
- Weight (a smooth piece of plastic wrapped around a heavy book)
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).
11. Washes. Wash 1-2 in 2XSSC/0.1% SDS for 20 mintues at room temp. Wash once in tube, then second in tupperware.
12. Wash 3 in .2X SSC/0.1% SDS for 20 minutes at room temp or at 55C.
13. Develop. Mount membrane on filter paper. Cover with saran wrap and pop it in film cassette with intensifying screen. Store at -80C ON (at least 15 hours)- may take longer for good exposure.
Solutions
Depurination
- 11mL conc. HCl 989mL dH2O
- store at RT for not more than one month
Denaturation
- 87.66g NaCl
- 20g NaOH
- ~800mL dH2O
- mix to dissolve
- bring to 1L final
- store at RT for up to 3 months
Neutralization
- 87.66g NaCl
- 60.5g Tris base
- ~800mL dH2O
- mix, pH to 7.5 with conc. HCl
- bring to 1L final
- store at RT for up to 3 months