Hi fellow researchers,
I'm getting quite experienced now in 16S amplification and sequencing, however, I'm having strange results here. I've performed a large number of 16S PCR using the same settings and I have a complete plate (96 wells) that have two strong band at ~500bp and ~700bp. I had in the past some samples showing these kind of double band but never a whole plate.
I tried reducing MgCl2, increasing KCl, adding DMSO, increasing annealing temperature, and all the common tricks to make a more specific PCR. None of this seems to work.
I'm getting to a point that this not artefact or unspecific PCR but something trully biologic, however, I can't work out why this plate is different from my over plates ...
Does any of you work that out ?
I do not have the time now to try to sequence the two bands apart and make a decision afterwards.
ps : plaque 2 is the one troubling me, the other is the one that worked pretty well. Acquisition settings are the same, the second one is the inverse image, but basically shows the same
Thanks a lot all
Are these from the same sample type (eg soil/saliva/feces etc)? Might try to reduce the number of PCR cycles, or use a different polymerase, or if nothing else works just cut the appropriate band from the gel w Pippinprep or similar.
Could be some sort of cross contamination. Run a positive and negative control DNA with similar PCR conditions and you might figure out from where the extra band is coming. Also it appears you are using too much of template DNA, its sitting in all your wells.
16S primers will amplify mitochondrial genomes & bacterial genomes and you can get some really unexpected products (e.g. fungal DNA when you expected nematode; human DNA when you expected fish, etc.). So, you are likely getting amplification from more than one type of organism. Human DNA is a common contaminant (from you). Depending on your project, you can either sequence a few of each band to see what is amplifying or just continue with the samples that give expected results.
Good luck!
Hi and thanks for your income.
Positives are good and negatives are also good.
I made the in silico PCR for mitochondrial genome amplification with my primer set and it returned nothing. I also performed PCR on human DNA and it gives nothing either.
I finally moved to the rest of the plates, from 6 96 plates only one is showing double band. It looks like something is weird with my second plate.
We will sequence the band appart to decipher what is going on in this samples.
I'll post updates here.
Thanks again
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