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?


Thank you!!!

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