Hi all,
This may be a stupid question but I want to generate a library using error prone PCR. Obviously I know the starting sequence but as the libraries can be completely randomised I'm not sure what primers to use to clone them into an expression vector. Do I just use the original primers and hope that sufficient primer binding occurs to yield cloning.
Best regards
John
Hi John,
If you don't mind losing some variants with mutations in the primer-annealing regions, then your way works. Otherwise, if your whole PCR product has to have mutations, you can clone the whole construct with TA cloning and then amplify from the plasmid.
Kamila
Hi John,
I don´t know about your specific setup but I guess you want to create a library of protein variants - so generally you shold aim to keep the exchange rate to 1-3 bp per kb template, because with high mutation rates you also accumulate high loss of function rates due to premature stop codons and deleterious aa exchanges.
So even if some rare exchanges within your primer binding sequence within the terminal gene region occur, your primers should still be binding to the template DNA allowing for moderate mismatches.
I´ve been using erPCR (2 ng template DNA) for enzyme library creation with MnCl2 concentration ranging from 0.01 to 0.5 mM and did not have problems with amplification and restriction/ligation based cloning afterwards (Golden Gate).
There is also a lot of protocols/literature available out there that also might help to help you setting up some trial runs
Hope that helps :)
Best,
Pascal
The main concept behind erPCR is use of DNA polymerase that has lower rate of proofreading the template DNA or one can say higher rate of incorporating wrong bases into the growing DNA. This procedure generally excludes the primer:DNA complex. The errors in gene or amplifying region are to be found within the primer sequence. This method is used to study the effect substitution of various amino acid or nucleotides in a sequence.When studying variant proteins we call it as directed evolution.
I believe that even if you incorporate desired restriction sites in your primers that will not alter the result after carefully designing them.
Best wishes
Asif
- Badges
- Science method