Every time we run the PCR, Especially for protein expression etc, we fine mutations in out fragment which dont let our protein to be expressed. What are the ways, conditions, chemicals to avoid any mutation in the fragment. Thanks for your reply..
make your primers for PCR anneal at close to their extension temperature of the enzyme( 68 or 72c) . enzymes work at all temperatures but make more mistakes at low temperatures while the primer is annealing and the enzyme is starting to extend. Use a high fidelity enzyme with proof reading ability and do not run too many cycles of pcr before cloning
Hi,
Mutation is a result of errors exerted by DNA polymerase in PCR. To avoid this, I recommend you not to use common Taq Polymerase instead you can use high fidelity and hot start polymerase, which are able to amplify large fragments without errors and they just function exactly by initiation of PCR.
We use Phusion Taq as well, which is high fidelity (but more expensive). NEB claims a fidelity 50x that of standard isolates. Also of course minimizing cycles will minimize error, as with each additional cycle you will increase the amount of error per the error rate of the enzyme, as well as propagating (at an exponential rate) errors which are already present from previous cycles. Others have already said these things, but I thought this might be of interest to you: https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/pcr-fidelity-calculator.html
It will allow you to calculate the expected error in your PCR. As you can see increasing enzyme fidelity will decrease error, and decreasing number of cycles will also decrease error.
use of phusion or q5 taq will solve the problem related to mutations. Strictly follow annealing and extension tempreatures.
Good luck
i am using Pfu enzyme, even then there are errors, at least 1 error/kb. it is affecting my protein, it have proof reading ability, even then leaves an error.
Use high fidelity proof reading DNA Polymerase like KOD or XT5 Polymerase.
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