I have done a 16S rDNA amplification of a metagenomic sample. I got a clear band near 1.5kb in the gel. Later on purification and again when we do the same PCR with the exact same primers and other components, i am not getting any amplification. The yield after purification is also good.
Can someone help me with this issue ??!
Your question is very confusing.
" Later on purification and again when we do the same PCR with the exact same primers and other components, i am not getting any amplification. The yield after purification is also good. "
- I did not completely understand the above point, why do you want to do PCR on purified product?
- What is you aim? What do you mean by other components?
Hello Abhijeet Singh
So, basically we gave the amplicon for sanger sequencing after 16S PCR. But turns out, the sequencing peoples are saying that they aren't able to sequence it as they also need to amplify at the initial stage.
to verify this issue we did another PCR with the same product.
Other components means the Taq polymerase, dNTPs, Buffer, Primer etc.
Dear Sarath
You mentioned that the sample you analysed is a metagenomic sample an you attempted a Sanger sequencing methodology. But, however Danger sequencing is not suitable for metagenome sample. As you get mixed peaks with a template extracted from microbial colonies of multiple origin.
What is your PCR amplicon size? Which region of 16S rRNA did you target? Did u quantify your DNA eluate? If you are planning for metagenomic analysis I would prefer Ilumina sequencing platform targeting V3 V4 region. Please go through the attachment.
hello Yugendran T
thank you for your valuable suggestion.
but, i dont understand one thing, why is the amplicon that has been amplified once is not getting amplified when we do the same PCR again.
and can you please explain the region of 16S target that you were discussing about.
thank you
Sarath Nair
The reason why I was talking about the regions within the 16S ribosomal sequence was because there multiple universal primer pairs available. Hence, wanted to know about the primer pairs that you have put into use. The bacterial 16S gene contains nine hypervariable regions V1-V9, and of these the semi-conserved hypervariable regions V3 - V4 is extensively targeted for bacterial identification, as it can provide resolution at the phylum level as accurately as the full-length 16S gene.
The are multiple reasons for the non-reproducibility of your previous PCR assay. First of all what is the amplicon size for your primer pairs. Is 1.5 Kb within range of your amplicon size? Are these primer pairs in-house designed or previously published one? Have you carried out BLAST analysis for your primer sequences and do you have the sequence specificity data for it?
Secondly what was your thermocycling condition? If you have the previously extracted template DNA then please try setting up a gradient PCR across 50 - 60
˚C and let me know the assay outcome. The I would be in a position to comment on the outcome of your previous PCR assay.
Otherwise with the data you have provided things boil down to primer pairs and the template DNA. Have you quantified the DNA that you had extracted for the second time. Was the yield good?
Thanks
Yugendran
Yugendran T
i am sorry, i made a small mistake in the question.
this is a metagenomic sample, but the DNA wasn't from the entire soil. it was first cultured and the bacteria was isolated. then the DNA of the bacteria was isolated which was further proceeded with 16S PCR.
The yield after purification is good. it is 43ug/ml.
The primer used was taken from a publication.
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