RAPID BIDIRECTIONAL SOUTHERN BLOT FOR CLONED DNA
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A. Process gel
1. Electrophorese sample in agarose gel, 0.5 to 1 cm thick, 0.5-1.2%.
2. Expose gel to UV light for 1 min on 265 nM light box (or depurinate in 2 vol of 0.25M HCl for 2X15', and rinse in dH20).
3. Break & denature DNA with 2 vol of 0.5M NaOH/1.5M NaCl for 2X 15'.
B. Transfer
1. Saturate in 0.5M NaOH/1.5M NaCl: 2 sheets of nytran and 6 sheets of 3MM cut to the size of the gel.
2. Make a sandwich: 3 sheets of wetted 3MM paper with 1 sheet of NC on top. Place gel on top of this. Add another nytran sheet, then 3 more 3MM sheets.
3. Place a 2 inch layer of paper towels beneath and above the sandwich. Add a weight to ensure even contact.
4. Transfer at room temp for >1 hr.
5. Rinse nytran 2 min in 2XSSPE. UV crosslink in a Stratalinker, (or dry by baking 30 min - 2 hr at 80℃ a vacuum oven.)
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1. 0.25M HCl 1 gel
0.25M HCl 20.84 ml conc. HCl (12M)
H20 to 1000 ml
2. 0.5N NaOH/1.5M NaCl 1 gel
0.5N NaOH 50 ml 10N
1.5M NaCl 87.66 g solid
H20 to 1000 ml
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Comments:
Large fragments (>~5 kb) don't transfer very well by this method, so expect weaker hybridization signals for these large fragments than you will get for smaller ones.
Must use nytran for this alkaline transfer method - nitrocellulose won't work.
