Southern Blotting using Stratagene's PosiBlot
Dominik Haudenschild, May 1995 at Genzyme
For gels at most 1% agarose, about 0.7cm thick
• Depurination:
-soak gels in 0.25N HCl for 20 minutes with gentle shaking
-the bromophenol blue dye should turn green with HCl treatment
• Denatureation:
-soak gel in 0.5N NaOH / 1.5M NaCl for 30 to 45 minutes, shaking
-the bromophenol blue should once again turn blue
• Neutralization:
-soak gel in 1M Tris pH 7.5 / 1.5M NaCl for 30 to 45 minutes
• Assemble in PosiBlot apparatus as in Stratagene's instructions
• Blot for 30 to 60 minutes at 75mm Hg pressure
• UV crosslink the DNA onto membrane using Stratalinker on AutoCrosslink setting
Random Prime Labelling Using Stratagene's PrimeIt II kit
Generating random primed, double stranded labeled probe
• add 25ng DNA template
• bring volume to 25µl with water
• add 10µl random oligo primers
• boil at 95°C for five minutes then quickly spin reaction to bottom of tube
• add 10µl of 5x primer buffer
• add 5µl of labeled nucleotide (3000 to 6000Ci/mmol)
• 1µl Exo(-) Klenow enzyme at 5 U/ml
• mix thoroughly with pipet tip
• incubate 37 to 40°C for 2 to 10 minutes
• add 2µl stop mix
* optional: clean probe over desalting column
RapidHyb protocol
Using random primed double stranded DNA probe (1/2 of 25ng reaction = 2ng/ml)
or RNA probes (1/2 of standard reaction = 6ng/ml)
• Prehyb HyBond N+ for 15 minutes in RapidHyb at 65°C (70°C for RNA probes)
If using roller bottle in hyb oven, use at least 10ml
• Boil probe for 2-5 minutes at 95°C to 100°C
• Snap-cool probe on ice
• Add 1ml prehyb solution to probe, then add probe to membranes
• Hybridize at 65°C (70° for RNA probes) for 1 to 2 hours
Overnight is OK but background increases
• Wash once 20 minutes at room temerature with 5x SSC + 0.1% SDS
• Wash twice 15 minutes at 55°C to 65°C with (1 to 0.1)x SSC + 0.1% SDS
• Wrap in Saran wrap and expose to film overnight