When amplifying SARS-CoV-2 genome in amplicons by the use of ARTIC network primers (v4.1) sometimes I get double band: one band at 400-500 bp (which is the amplicon length) and another one at 600-700 bp. Negative controls are clean. I have already checked if primers might be binding another region, but they don't.
Actually this only happents sometimes, not always. Any idea of what is amplifying at 600-700bp?
There can be many reasons for non-specific amplification.
If it only happens sometimes as you say, you may want to check/calibrate your PCR machine as some wells may work better than others.
Other than that, a quick PCR optimization may help, run a temperature gradient and see if that solves the problem
Hi Carolina
it has been proven that samples coming from infected individuals do not contain only one clone from the COV19, but Melting of some clones with different genomes (light differences of course, just due to high copy rate and error under-regulations). in this respect, having more than one template can led to your results.
fred
Péter Gyarmati Melting temperature for annealing is standarized at 63 ºC by ARTIC network because we perform a Multiplex PCR with several primers for different amplicons in one tube, therefore it is supposed to work... Anyway, I will check it out just in case... Thanks for the input!
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