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non specific amplification


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#1 maxmix

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Posted 22 February 2010 - 01:35 AM

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

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#2 Clare

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Posted 22 February 2010 - 04:14 AM

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

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






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