I performed 16S rRNA PCR Analysis on a total of 6 samples. I used universal primers 27F and 1492R for this PCR analysis. 5 out of 6 samples show bands detected on 1500 bp marker, however there is one sample shows band on 10,000 bp marker. Is that means that the primers didn't read the sequences of DNA template?
I used 5ul DNA template for a total of 15ul for each sample. What should I do to troubleshoot this problem? I couldn't send the sample for sequencing since it does not show a perfect band on gel electrophoresis.
Do help me for this. Thank you.
Did you put 5 ul of template DNA in 15 ul PCR mixture?
If so, I think you DNA is excess which leads to non specific amplification.
The other 5 samples according to you gave bands at 1500 bp and only one gave at 10000 bp. So, I assume the bands at 1500 are the desired ones and the one at 10000 is the problematic
Did you check the DNA concentration and purity by spectro or running on gel? In my opinion, I think the DNA of your other 5 samples are too dilute. So, when you put considerably higher volume of template DNA, its final concentration in PCR mixture become optimum for DNA amplification.
In the sample no 6, the DNA is really concentrated ( just opinion). So, when you put 5 ul in 15 ul PCR mixture, the final conc of template DNA becomes high leading to non specific amplifications, which I think might be responsible for the 10000 bp band.
Or 10000 bp band is not an amplicon, its rather the DNA band because amplification might not have take place due to some other factors.
As for sequencing, you can send first some of your extracted DNA for sequencing rather than your amplicon.
I
- Badges
- Science method