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Low blotting efficiency (southern blotting)


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#1 TCT

TCT

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Posted 31 March 2005 - 04:26 AM

Hey


I have a problem with getting all of my DNA (genomic and plasmid mix) transfered from my 0.7% agarose gel (SeaKem LE) to my membrane (Nytran N membrane, Schleicher & Schuell...)

The total DNA is digested with Hind III before electroforesis, and the gel is run at 80 V for 3 hours. The gel has been depurinated with 0,25M HCl in about 10 min, before denaturation and neutralization. The blotting procedure are performed with a turbo blotting system and with 20x SSC as transfer buffer. Blotting time that has been tried out is 12 hours, 46 hours.
12 hours gives a poor transfer, while 46h gives a bit better transfer. How can I increase the transfer?

I have tried a alkaline transfer, but that doesn`t give any better result.




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