Hi,
I am having hard time with the PCR of bacterial v1-v9 region using 27f and 1492r primers on DNA isolated from PBMC. I have tried various conditions of PCR and concentration of primers and template but the problem persist. I am using KAPA HiFi HotStart ReadyMix. Can you help me resolve the issues. I am uploading an image of gradient PCR FROM 48-62 °C for reference.
Hi Mohd,
typiccally when seeing the gel you have unspecific bands (due to the choice of the primers, quality of samples, multiples transcripts from the same gene....)..
the problem could also be that you use bacterial primers on PBMC (human I guess), genomes and sequences can differ, or you're targeting the bacterial sequence in the blood...I don't know.
I would do the same thing but between 60 and 65, one degree by one, adding some DMSO at 5% in the solution.
all the best
fred
Hi Frederic, thank you for your response. I trying to quantify bacterial DNA that might be present in the pbmc from human sample. I am using the 27f and 1492R primers. The DNA yield is good. I am trying reduce the multiple bands that I am getting as shown in the figure.
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