Assembling ePCR library using Gibson assembly or Golden Gate assembly?
Currently I am using a GeneMorph kit to generate error prone PCR libraries of a gene region. Since the region I am using is internal to the gene I am not using restriction sites. In this case I wanted to use Gibson assembly or GoldenGate assembly to clone the ePCR fragments back into the vector. However, the workflow I employ to do this is 1. Use ePCR to generate gene libraries 2. Use PCR to add Gibson overhangs or type II restriction sites to the gene for GoldenGate assembly, 3. Use PCR to linearise the vector and finally, 4. Use Gibson or golden gate assembly to clone the genes into the vector.
With this in mind I wanted to ask is this workflow a sensible approach? Secondly, I wanted to ask how much of the ePCR product (in volume) should I add to the second PCR step where I add the Gibson and Goldengate overhangs? Finally, would it be sensible to generate the Gibson and GoldenGate overhangs during the ePCR step?
Thanks in advance
John
It seems like Golden-gate might chop off some PCR fragments carrying cutting sites because they can be generated in the gene during ep-PCR.
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