I need help with a PCR. I have the following primers mecA-F 5′AAAATCGATGGTAAAGGTTGG-3′ / mecA-R 5′-AGTTCTGGCACTACCGGATTTTGC-3′.
I made an initial PCR with 12.5 of GoTaq Green Master Mix, 2x, 0.8 of each primer, 5.50 of PCR-grade water, and 3 microliters of the sample, based on the article "Characterization of Staphylococcus aureus isolated from chicken and quail eggshell."
The PCR conditions were as follows: 5 min at 95°C, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 45 sec, and extension at 72°C for 1 min. The final extension was set at 72°C for 10 min.
I tried three different annealing temperatures, but it didn't work. What should I do next? Should I continue with the same primer concentration, or should I increase the annealing time and the number of cycles?
What was the concentration of primers and templates used? We need optimum concentrations. Too much and too little can create problems.
Typically, genomic DNA is used at concentrations of 10 ng to 500 ng per reaction. Plasmid DNA templates are usually used at concentrations of 1 ng to 100 ng per reaction. Primer concentrations typically range from 0.1 μM to 1 μM each.
we need to re-check the melting temperature of the Primers(both strands) and annealing temperature, best to go for gradient PCR.
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