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PCR band for water but no contamination, Digestion not working - (Sep/28/2006 )

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I thought my primers were contaminated and got a new set fo primers from the company. Again, the same problem. So contamination in the primers is highly unlikely.
As you said, maybe one of the other reagents has some small contamination which is amplified only by this set of primers.
I guess I have to try with fresh stocks of the other reagents.

Would it help if I increased the annealing temperature? According to literature, the annealing temp. used for my primers was 66 degrees. But I see nothing at 66, and I can see bands only at 62 degrees.

Any thoughts?
Thanks for your help.

QUOTE (gsamsa @ Oct 5 2006, 07:56 AM)
QUOTE (genomicsgoof @ Oct 4 2006, 11:09 PM)

Yes the other components are clean, since they have worked in other PCR's.

Have you consider that one of your reagents may be contaminated with a really tiny amount of DNA (1ng)?

If that's the case, it may be that only those primers amplify that sequence enough to be seen in a gel and the other primers don't bind to that sequence in the rest of the PCR.

About cheking your primers that way, wouldn't it be easier just run 20ul of your stock solution in a gel and see if there is a extra band? Don't you use working concentrations of your primers? What I do if I get contamination is first get rid of all the working concentrations used, make new ones and perform the same PCR. It usually works most of the times.


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