I would like to know how double stranded DNA becomes single stranded when binding to nitrocellulose membrane in southern blots. Does it require a special reagent? Is denaturing a special property of the membrane itself?
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2 Answers
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The DNA is denatured in-gel prior to transfer using alkali conditions, most commonly a solution containing 0.5 M NaOH (buffered with 1.5 M NaCl). Following that the pH of the gel is neutralised in 0.5 M Tris-HCl, pH 7.5; 1.5 M NaCl.
I have found the Roche DIG application manual to be a well-written and reliable resource for performing Southern and Northern blots. The section on Southern blots starts at page 94 and you can modify the protocol to suit other detection systems.
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No, there is no such property present in membrane. After transferring to membrane they treat with alkaline solution to denture dsDNA to ssDNA for binding of probe.
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1$\begingroup$ Don't most transfer methods only allow ssDNA to transfer from the gel to the membrane? $\endgroup$ Commented Sep 29, 2016 at 5:08