Help! Problem about Southern Blotting

John Watson watson_j at bms.com
Wed Jul 9 15:27:21 EST 1997


Wilson wrote:
> 
> Dear all,
>         I have found a problem in the Southern Blotting.  All digested
> genomic DNA (size from 11kb to 0.5kb) and lambda DNA marker still stayed
> in the agarose gel after overnight capillary transfer. While the
> bromopheonl blue dye have successfully transfered to the membrane.
> 
>         I have used 10X SSC as transfer buffer and the treatment of the
> gel was the incubation with agitation 0.4N NaOH with 0.6M NaCl for 30
> minutes and then 0.5M Tris-HCl pH 7.6 with 1.5M NaCl for another 30
> minutes.  The methods have been used in our lab without any problems.  I
> even my boss don't know what wrong in the blotting.  Please help me to
> solve the problem.  Thank you very much.
> 
> Wilson

Try including  acid depurination step -- soak the gel in 0.25 M HCl
until the bromphenol blue turns a yellowish-brown color - usually about
10-15 minutes. Then denature and neutralize as usual. There are also
protocols that use UV light to fragment HMW DNA prior to transfer, but I
am not familiar with those.  You might try Sambrook et al. or the Red
Book if you are interested.

AJW
----------------
John Watson
Bristol-Myers Squibb Co.
watson_j at bms.com
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