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
-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
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-