I'm analizing bacterial 16S sequence and having some trouble with the PCR reaction. The genomic DNA integrity is ok as shown in the agarose gel, but the PCR products are somehow deteriorated and appear as a comet.
We've been using the same protocol for some time now and had no problem until now. Any ideas on what could be happening?
If this pcr has been working for a long time them I think that you may have dissolved the primers in water. Absorbing CO2 from the air makes the water acidic and depurinates the primers and they fakll apart. Buy new primers and dissolve and store in TE. The tris buffers the primer alkaline and the EDTA extracts Mg so DNASEs can not work to degrade the primer.
Looks like DNAse activity (which you have ruled out) or mispriming due to low primer specificity/secondary structure of template and/or primers. Might be DNA contamination as well. If the same protocol worked before, I would suggest making fresh stock solutions of everything. Then, try increasing annealing temperature by 2-5C /increasing initial denaturation temperature to 95-97C and cycling denaturation time to 15-30s.
If this pcr has been working for a long time them I think that you may have dissolved the primers in water. Absorbing CO2 from the air makes the water acidic and depurinates the primers and they fakll apart. Buy new primers and dissolve and store in TE. The tris buffers the primer alkaline and the EDTA extracts Mg so DNASEs can not work to degrade the primer.
The origin from the DNA is the same? Did you try to add in the PCR mix BSA? Claudia Hoepfner
Paul Rutland that was the problem indeed. We prepared a new set of primers and the reaction went just fine. Also asked what was used to dissolve and store the old ones and told me they used water. Thank you for the great explanation!!
very happy that your problem is solved Claudia Hoepfner and thank you for letting me know. all the best Paul
In my experience, I dissolve the stock in TE buffer and the dilution in water (because more time I need for the sequencing).
I agree with Paul .One of my students faced the same problem previously when he diluted the primers with water and also the problem was solved by using the TE dilution buffer for working primer solution as it stabilizes more the primers.
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