Alkaline downward trnasfering

Malay curiouser at ccmb.ap.nic.in
Mon Mar 8 03:04:38 EST 1999



>I use alkaline downward transfering method to transfer DNA to nylon N+
>memberane. After depurinization in 0.25 N HCl for 15 min, I just rinse
>the gel with D.W. instead of denaturation in o.4 N NaOH , then the gel
>was transfered with 0.4 N NaOH for several hours. I do not know if the
>DNAs in my gel have been changed into single strand during transfering.
>is it necessary to denature the DNA before transfering since the
>transfer buffer is 0.4 N NaOH?  Thanks!
>
>Yang
>the University of Northe Carolina at Chapel Hill.

Dear Yang:

I don't know what are the band sizes of the DNA you are transferring, but
from my experience I can say that upto ~20 kb band can be transferred
without doing anything! Not even depurinization. Just run the gel and set
the transfer using 0.4N NaOH. You don't even need to fix the DNA with the
membrane after the transfer, if you are using N+ from Amersham and alkaline
transfer. Some positively charge membrane manufacturers don't recommend
alkaline transfer. Just check once the recommended protocol.

Malay

Malay Kumar Basu
Centre for Cellular and Molecular Biology
Hyderabad 500007
I N D I A

Fax: (00-91)40-7171195
Phone: (00-91)40-7172241
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curiouser at ccmb.ap.nic.in






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