why this happens ?? I have tried Q5 and phusion polymerase also but didn't get the good result. I think i correctly design the primers. First pcr using vector or cultured broth give out pcr product but using these cannot show any pcr product.
I have never seen this situation before such as E. coli, Lactococcus, Ralstonia, Shewanella, Corynebacterium and so on. I had done many restriction cloning before using many microorganisms. This time bacterial strain is C.acetobutylicum atcc824.
questions are
1.are these polymerase choping off the end of the pcr product?
So Nested pcr is required?
2. Template is somehow damaged? I have cloned only one gene but next one i have failed.
I already tried DMSO or GC enhancer and this organism's genome is i think rich in AT rather than GC and found not working
what do you mean by "does not work" is there no product at all or is there a smear of amplified product of higher size than the expected amplimer?
Nested pcr is a good idea and it works well but if you are getting a smear then you are probably over amplifying. A second round amplification should be on 1ul of 1/100 dilution of first round product.and only between 10 and 20 cycles. Are your negative control samples clean of amplified product?
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