Hi everyone.
I have a protocol that generates antibody variable regions from B cell cDNA through nested PCR..
It uses the cDNA as template for a nested PCR reaction series where I amplify heavy and light chain variable regions in separate reactions. I use multiple forward primers (to find as many families as possible) and a single reverse primers for each PCR step.
I have previously successfully completed these reactions multiple times where the final step generates a series of nice, clean band in the 400 bp range when visualized on a gel (Image 1).
Since a couple of months my PCRs are total failures and I can't understand why. The final PCR products are only a smear (image 2 and 3) with weak to non-existing bands. From having nice, clean bands with an around 30% positive hits I get weak, hardly defined bands in a smear and a 2-5% yield.
The protocol is the same, Ive done the following:
- Changed the polymerase to new batch - (no difference)
- New water - (no difference)
- Re-ordered primers - (no difference)
- +/- cDNA amount - (no difference)
- +/- Tm 2°C - (no difference)
I went back to old cDNA that I previously successfully generated nice bands from and now I only get a smear from that material as well....
When doing the light chain reaction with the new cDNA I can still generate nice clean bands (image 4) so the reagents, polymerase, cDNA and thermocycler must work at least semi-correct I assume.
The only thing I have left is the primers but I can't understand why they just would stop working, especially since I ordered them fresh twice now and still can't get the reaction to work. The sequences haven't changed so why would they bind in my first experiments but not now?
Since I don't get enough material to visualize on a gel from the first nested PCR I can't pinpoint if the problem lies in the first PCR or the second.
Anyone here that have experience with troubleshooting PCR, especially nested PCR that have any advice on what my problem could be and/or suggestions what and how to continue my troubleshooting?
Thanks in advance for any suggestions!
It looks like you have post pcr product contamination leading to great over amplification when the product anneals with other product molecules to form amplimer smearing. Run a pcr with water instead of dna and if you get smears ( or bands) in these negative controls then either your working area or reagents are contaminated and this is showing in your pcrs
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