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.
Dear Syafiqah Mhd Jamal,
With your question before troubleshooting would like to know?.
1. Source of Parental DNA for all the sample, is it same?
2. Ammount of DNA used in each PCR reaction in terms of concentration? (estimate DNA on nanodrop)
3. 10,000 bp Amplicon, Unused Primer Dimer Status?, (Yes, primers didn't read the sequences of DNA template?, Can be, go for sequencing using Forward Primers)
4. DNA marker maximum band size on gel in KB.
Troubleshooting : Repeat PCR with known templet concentration, elute DNA and send for sequencing.
All the Best
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