weird site directed mutagenesis products - (Mar/12/2010 )
I recently performed site directed mutagenesis introducing an XbaI site into a 8.5 kb plasmid. I used a protocol comparable to stratagene. The primers I used are only 29% GC (cant get better). Anyhow I restriction digest screened 16 products (correct product gives 3 bands on gel). Four of the 16 were correct, however eight of them gave a weird 4 band pattern which sizes do not add up to original plasmid. I was wondering if anyone has had a similar experience? Should I just ignore those weird products and continue on and sequence the four correct ones?
You might have different fragments of almost the same size after digest. They would overlap and you wouldn't see them on the gel. I'd recommend not to bother and sequence your correct ones.
I think I will do that. Thanks!
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