Depurination for DNA blotting - (Oct/08/2006 )
Hi everyone,
When doing a southern blot, one of the steps is depurination (usually in HCL 0.25N) to make the transfer of the large DNA fragments easier. What about the time when we want to blot short segments of DNA, say less than 10 kb? Should we still do the depurination? Doesn't it harm the fragments breaking them down to small pieces? Should I simply omit the depurination step of the blotting? Please help me on this.
Regards,
Zaanaa
PS. Actually, I am blotting digested cosmids. So I have both over 10kb and below 10kb fragments.
-zaanaa-
QUOTE (zaanaa @ Oct 8 2006, 08:03 AM)
Hi everyone,
When doing a southern blot, one of the steps is depurination (usually in HCL 0.25N) to make the transfer of the large DNA fragments easier. What about the time when we want to blot short segments of DNA, say less than 10 kb? Should we still do the depurination? Doesn't it harm the fragments breaking them down to small pieces? Should I simply omit the depurination step of the blotting? Please help me on this.
Regards,
Zaanaa
PS. Actually, I am blotting digested cosmids. So I have both over 10kb and below 10kb fragments.
When doing a southern blot, one of the steps is depurination (usually in HCL 0.25N) to make the transfer of the large DNA fragments easier. What about the time when we want to blot short segments of DNA, say less than 10 kb? Should we still do the depurination? Doesn't it harm the fragments breaking them down to small pieces? Should I simply omit the depurination step of the blotting? Please help me on this.
Regards,
Zaanaa
PS. Actually, I am blotting digested cosmids. So I have both over 10kb and below 10kb fragments.
For fragments below 10 kb, depurination is unnecessary. For fragments over 10 kb, depurination is helpful. You should treat the gel with 0.25 M HCl no more than 10 min according to a manufactor's instruction of ROCHE. The bromophenol blue will be changed from blue to yellow during depurination, it is an indication of success of depurination and is always less than 10 min.
By the way, cosmid southern blotting is much easier than genomic southern blotting, omit the depurination step or not, in my opinion, doens't affect the final result.
-zhongmindai-