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non specific amplification - DNA amplification by PCR (Feb/22/2010 )

Iam trying to optimize Master mix to use it to amplify DNA to get 107bp with high G,C Content primer:
the forward primer: 5 AGCCAGTGCGCTTACCTGCCAGAC-3
and the reverse primer: 3-CATGGGTACGCGGCCTGGTGCTGCAGTGC- 5. (Garovic et al.2002),
i got nonspecific product as shown in the pic but the negative control is OK,,no product.
i used: 20pmol each primer, 1.5Mm Mgcl, 0.2 taq pol., DMSO=10%, DNA temp~250ng,
denatur, 94c for 30sec, annealing,61c for 30sec,,,exten=70 for 30sec.
tq
Attached Image

-maxmix-

What genome are you using?

I tried mouse, human and rat (UCSC primer search) and got no matches to the genome using your primer set.

Clare

maxmix on Feb 22 2010, 09:35 AM said:

Iam trying to optimize Master mix to use it to amplify DNA to get 107bp with high G,C Content primer:
the forward primer: 5 AGCCAGTGCGCTTACCTGCCAGAC-3
and the reverse primer: 3-CATGGGTACGCGGCCTGGTGCTGCAGTGC- 5. (Garovic et al.2002),
i got nonspecific product as shown in the pic but the negative control is OK,,no product.
i used: 20pmol each primer, 1.5Mm Mgcl, 0.2 taq pol., DMSO=10%, DNA temp~250ng,
denatur, 94c for 30sec, annealing,61c for 30sec,,,exten=70 for 30sec.
tq

-Clare-