I am a beginner of cDNA cloning for overexpression of my protein in cell line. I have extracted mRNA from cells, converted it into cDNA using Quantitect reverse transcription kit (Qiagen) and stored in -20. After that I have used this cDNA for Phusion PCR amplification with my cloning primers which have restriction sites and FLAG tag sequence. But still I have seen only primer dimer in gel , not my desired product. I have tried many times, changed different parameters but it does not show any positive result. In Real time PCR, I have used this cDNA and got ct value of 25 for my target gene and for Phusion PCR , I have taken 84 ng of cDNA.Is there any problem in my cDNA or my procedure?
Hoping for early reply
Thanking you
Try using basic Taq for PCR as this will often work when hi-fi enzymes don't. Of course, you may introduce errors, but at least this is a test of the cDNA. Gradient PCR on the annealing step can also help sometimes. Also, sometimes a primer just won't work, so it can be worth trying alternatives.
I agree it would be good if you optimize PCR using taq over a range of temperatures according to Tm of primers and also run some positive control to check if cDNA is not degraded. You can try different dilutions of cDNA as well.
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