Hello all
I am using double joint PCR method to generate mutant. I got correct size fragments of target and neo marker gene from first round of PCR. PCR product obtained from this were purified using gel extraction method and then equimolar amount (approx 25 ng each fragment) of both fragments were used for double joint PCR. But I am getting only smear. I am using correct primer. I tried with different brands of Taq polymerase also but same result. I used same annealing temperature as I used in first round of PCR (55°C). I am attaching gel pictures for both first round PCR and double joint PCR. Please suggest me.
Thank you in advance
Hi,
In my opinion you may have to work on optimizing the second PCR conditions, particulary annealing temperature, lower number of cycles and less template
Usually i use a more simple procedure. I mix 1 ul of each PCR product from the 1er round in 500 ul of water and use 1 ul of this mix for the second reaction. It's easier and I get PCR in the correct size. If you extracted PCR products from agarose gel it could contain PCR inhibitors. Besides that, try to reduce the amount of primers and increase the annealing temperature
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