When i did PCR for a house keeping gene for different isolate of Acinetobacter baumannii i got no band and a less band for two isolates. Then i did a gradient PCR to get intense band on both. Since the melting temperature of primer don't change, why do i get band only at different annealing temperature?
1.Is it due to some inhibitor present in that isolate?
2.is it that the melting temperature of that isolate differ from other isolate?
If iam wrong in my logic please guide me. Thank you.
Hi Nithin,
Monovalent, divalent, and polyvalent cations are known to affect Tm. I wonder the variation you have noticed is due to that. It is possible that different DNA templates contained varying concentrations of salts. How you have prepared the DNA template?
In addition to that, nucleotide mismatch (among various isolates???) may also affect the Tm. It is reported that a single mismatch can cause Tm to vary between 1 and 18°C.
Thank you Syed. I used crude extract as DNA template. I didn't know a single mismatch can alter the Tm to a wide extent. Normally all my isolate picked the gene at 55 c but one isolate picked at 59 c. And how do i know it is a mismatch?
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