Hi all,
I am carrying out an error prone PCR (ePCR) on a region internal to a gene that I cannot use restriction sites for. Therefore I cannot use restriction cloning to construct the library. I was wondering could I use Gibson assembly for this instead? Obviously I am anticipating loosing some of the fragments as mutations will be introduced to the regions which bind to the Gibson primers. I was also thinking of adding the overlaps required for Gibson cloning during the ePCR step. Is this advisable?
Thanks in advance
John
Hello John Edward Simons
Gibson assembly is a good alternative when one doesn't want to use restriction enzymes. But kindly make note of the following while performing:
- Gibson primers should have a good homology to the PCR product. If not, it might be difficult to amplify certain products where mutations are present in the primer binding region. You may once check In-silico for homology of your primers randomly expected mutated products.
- I am not sure about adding overlaps during ePCR step.
- There are several methods that does not require exonucleases like certain gibson assembly procedures. Some examples include using topoisomerase techniques, SLIC etc.
- There is a method called Restriction-Free Cloning. Kindly read about it through this link: https://bmcbiotechnol.biomedcentral.com/articles/10.1186/s12896-017-0346-5
Further reading required? Try this: https://bitesizebio.com/23458/protocols-for-cloning-without-restriction-enzymes-or-ligases/
Feel free to ask in DMs on RG if any inputs are required.
Cheers!
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