Nucleotide excision repair

Chandrasekhar Gujuluva cgujuluv at
Sun Jul 23 21:56:40 EST 1995

Hi! I would like to know whether anyone has any idea on the following. I do in 
vitro DNA excision repair assay from mammalian cell nuclear extracts using UV 
mutaginised plasmid DNA as the substrate and alpha-p32-dATP. I run agarose gel 
of the plasmid (after phenol, chloroform extraction) and do autoradiogram to 
visualise the specific excision of UV damaged DNA and new repair synthesis of 
DNA. Here is my problem: I see not only incorporation of p32 label in the 
plasmid DNA but also annoying smear in front of the band. Sometimes even my 
plasmid seems to be stuck in the gel (0.8% agarose in 0.5x TBE) and does not 
seem to migrate more than a few cm in the gel in spite of prolonged 
electrophoresis (although the dye front moves!). Does the experts in this area 
have any idea on what is going on here? How could i get incorporation of label 
only in the plasmid DNA and no smear? I would appreciate your reply either to 
this newsgroup or to my e-mail box. Thanks.

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