I picked up a single colony of Salmonella Typhimurium with a gene specific primer but several times i have tried...but not getting any results.
So please suggest how can I troubleshoot this.
Thanku
It is really hard to diagnose your gel, it does look like the only thing you see is the primers at the bottom of the gel.
If you have some pure DNA as a control, that might be helpful to be sure that your primers and PCR conditions are appropriate for the amplicon.
I have frequently noticed with colony PCR that more template is not better. Sometimes when I have a colony PCR that fails, I will dilute the sample 5 or 10-fold and then it works. If you have too much gunk around it can be inhibitory. So give that a try.
Provided your PCR conditions and Primers are perfect, the only reason I can think of for this kind of result is DNA overdose that might have changed the pH of your mastermix despite buffering. To avoid this, pick a single colony and mix into 50 ul nuclease-free water. Boil the sample at 95 C for 10 min. Vortex thoroughly and pellet down all the debris. Take 2 ul of supernatant as DNA in your PCR. This will reduce initial dna input and will yield a better pcr product.
All the best
Siva
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