My opinion is that you should ALWAYS sequence PCR products if in the
end it will be important to have it error free (i.e. you are trying
to express active protein). Even with a high fidelity enzyme, there
can still be an errors . Sequencing now is a lot easier than doing a
bunch of experiments only to find out later that you had an error.
You can't rely on your polymerase having the fidelity to make an
error free fragment-- it will only reduce the likely hood of having
an error.
Mike
On Jan 18, 2010, at 9:07 AM, Azam Rahimpour wrote:
Hi
I need to amplify a 1.8 kb protein coding gene for cloning and
then expree it in mammalian cells, I used both pfu and long pcr mix
enzymes (fermentas). pfu amplification was failed but I had sharp
bands with long pcr mix, but I am still uncertain of feidelity of
that mix supplier says its error rate is 3 time less than tag, and
it means that there may be some errors, I dont know what to do
should I do more try with pfu or cloning 1.8 fragment amplified
with long mix is safe?
regards
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