nightingale, on Aug 18 2009, 01:39 AM, said:
Dear all,
a question that is on my mind
...
in the application in which :
do PCR,
gel extraction for the band,
sequencing
i already know the base sequence for my gene/mutated one from the PCR
so,,
why sequencing ( as if once again ?? ) ???
is it because of the amplification error ???
if this is the case, i can choose a high fidelity enzyme
which,i think, is cheeper than using a whole new device to know the sequence ...
my apologies, if my question seems silly ...
thanks for ur time
I've been using Phusion High Fidelity polymerase of late for cloning, and have gotten many many point mutations in my sequences, and the point mutations vary by individual colony, so it's not a mistake in the original sequence. I don't know if it's my polymerase or bad E. coli cells, but I now suspect the polymerase.
In the past (and in some instances currently) Phusion has given me no problems like this, so it may just be a bad lot of polymerase.
In either case, PCR is not always perfect, even if you have a high fidelity polymerase. You can have a bad lot of the polymerase, or it could have been left out of the freezer for too long by another lab member, and the enzyme could be affected.
Furthermore, if you're making a mutant, you better be sure it's what you think it is. A bad PCR product could knock a downstream gene out of frame (if it's a bacterial gene in an operon). If you're doing allelic exchange (homologous recombination), if you're sequence flanking your mutation is mutated, it could directly affect a upstream or downstream flanking gene, if that mutation is transferred.
To me, there's too much at risk to NOT sequence.
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