RAPID BIDIRECTIONAL SOUTHERN BLOT FOR CLONED DNA

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RAPID BIDIRECTIONAL SOUTHERN BLOT FOR CLONED DNA

RAPID BIDIRECTIONAL SOUTHERN BLOT FOR CLONED DNA
by Michael Koelle, modified from Ken Irvine

Link: http://www.med.yale.edu/mbb/koelle/

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° C a vacuum oven.)

------------------------------------------------------------------------

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

------------------------------------------------------------------------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.