Blotting DNA from polyacrylamide gels

[Audrey Ah Fong]

Hi ! I'm having probelms regarding transferring DNA from polyacrylamide 
gels.  I'm actually running DNA on DGGE gels (polaycrylamide with urea and 
formamide) and have to transfer DNA on nylon membranes.  The protocol I 
have, used electroblotting but after staining the gels, the DNA, especially 
the bigger fragments are still there.  I do not know what i could be doing 
wrong.  To tell more precisely what I'm doing, after running the gel I 
depurinate the gel for 5 minutes in 0.25 M HCL, rinsed in water followed by 
5 minutes in 0.5 M NaOH.  I do neutralise for 5 min in 0.5 M Tris-HCl and 
then equilibrate in transfer buffer before I do the blotting at 15 V 
overnight.  Do you think that i'm not soaking the gels longer in NaOH?  I'm 
kind of desperate and I hope you could help me or tell me what you have been 
doing to get good transfer.

Hoping that you could be of help.  Thanks in advance

Audrey

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