So, I have been trying to run a pACYC PCR which will be used later on for a Gibson Assembly. However the PCR is not working. I have already tried gradient PCR and changing extension time; however it has not work. I am currently preparing the following master mix:
20 ul of pACYC Fwd
20ul of pACYC Rev
8 ul of Template
200 ul of Q5 master mix
152 ul of water.
I am running 25ul in each PCR tube. When running the gel, what I see is a long smudge and no clear band. I also run the template alongside it and it worked perfectly fine.
Just to say, I have already used brand new diluted primers, different set of templates (from miniprep) and a new vial of Q5 but the final outcome was the same: no clear band.
Can you please give me any suggestions?
N.B.:
Thank you!!!
What weight of dna is there in 8ul of template and what size is your template and also the size of your target amplimer. What is the Tm of your primers and what annealing temperature and extension time in your pcr cycling
1. Try check the primers, check if the sequence are correct
2. Check the difference in melting temp of both primers, if the difference is too big, amplification may be affected (preferable difference within 5C)
3. Check the final concentration of template for your pcr. Normally
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