Tests for DNA contamination in reverse transcriptase PCR. (A) Common bean sequence characterized amplified repeat (SCAR) marker SK14, linked to the Ur-3 rust resistance locus. From our experiments, SK14 amplifies from genomic DNA but not from cDNA, presumably because SK14 is from the intronic region of the gene. Forward and reverse primers derived from the SK14 sequence were used to amplify a 600 bp product from genomic DNA and cDNA; no amplification from cDNA was observed. Lane 1, 100 bp ladder; Lane 2, genomic DNA; Lane 3, leaf cDNA; Lane 4. Negative cDNA control (no reverse transcriptase was added to cDNA synthesis reaction); Lane 5, H2O only control; Lane 6, 100 bp ladder. (B) Primers from contig32565, a sequence with homology to a MADS transcription factor amplified long flanking intronic genomic DNA yielding a 1200 bp amplicon from genomic DNA and a short 300 bp amplicon from cDNA. The order and contents of lanes 1 to 5 are identical to those in panel A.
Kalavacharla et al. BMC Plant Biology 2011 11:135 doi:10.1186/1471-2229-11-135