Dear Herman Silva and Diya Banerjee
We have been discouraged to learn that you are having problems with the
nga1139 primer pair. We designed the primers to try and avoid just these
sorts of problems.
The nga1139 primers we use are:
nga1139L TAG CCG GAT TTG GTA CC
nga1139R TTT TTC CTT GTG TTG TCC
These primers allow the amplification of a 114 bp product from Columbia
genomic DNA, and a slighty larger product from Landsberg erecta (118 bp or
so). We have not had problems amplifying from genomic DNAs (Col, Ler,
Nd-O, No-O, WS, RLD, all 300 members of the RI line mapping population),
although resolving the polymorphisms does require special gels (3% MetaPhor
We use no special PCR conditions. Genomic DNA, primer, and enzyme
concentrations are standard. Mg++ is at 2.5 mM. Our cycling protocol is
based on 20 ul reactions performed in thin well microtiter plates in a MJ
Reseach DNA Engine. We perform 30 cycles of 15 seconds at 94 C, 15 seconds
at 55 C, and 30 seconds at 72 C; then hold at 4 C.
I hope this information may be of use for you.
Arabidopsis thaliana Genome Center
University of Pennsylvania
Philadelphia PA 19104-6018