Hello everyone!
I have a big problem with my qPCR SYBR Green I (see the following file). I thought I have the ''Hook effect'', but It seem that is only with probes... So I really don't know how to explain my results.
I test five times with different parameters and I still have those results. I change the hybridyzation temperature, final DNA concentration, final MgCl2 concentration, different DNA extract and final primer concentration. I'm working with environmental samples, and I know that PCR inhibition could be a problem. Then, I test PCR inhibition with an IAC (internal control amplification) and with a serial dilution of my DNA extracts. No anormal inhibition of my PCR. The most ''bizarre'' is that my swine DNA extracts have a very nice exponential qPCR amplification curve, but not those of soil.
I look forward to your comments, suggestions and explanations.
Are you having evaporation issues with those samples? Slide 37 of this presentation sort of looks like your curve.
Good luck, I look forward to seeing the resolution, weird yet interesting problem.
No I don't. I add 25 uL total for each reaction and I had 25 uL after my qPCR.
Thank you for your help!
I found this article about the benefits of adding BSA to PCR reactions, it binds inhibitors and makes the enzymes more thermostable... don't know if it will help if you are using a manufacturers kit....
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3466135/
its interesting though. Sorry I can't be of more help. Hope you find a solution.
- Badges
- Science method