cloning the wrong way?

Chris Boyd cboyd at holyrood.ed.ac.uk
Mon Mar 6 08:49:04 EST 2000


Chris LaRosa <plarosa1 at navix.net> wrote:
: Alternatively,,,,use  error correcting polymerases.


: "Richard P. Grant" wrote:

:> In article <38AAD3B4.D212C32C at uni-konstanz.de>,
:> Frank.Fackelmayer at uni-konstanz.de wrote:
:>
:> > ALWAYS!!! Especially when using Taq polymerase which is notorious for
:> > making mistakes.
:>
:> Pish.
:>
:> To the second part of this, anyway.  Drop the dNTP conc to 50 uM (from
:> 200 uM) and watch your error rate fall away.
:>

Hmm, I missed the early part of this thread, but can guess the context. 
I agree with Frank: ALWAYS sequence inserts that have been PCR cloned. 
We have found errors even with the use of proof-reading polymerases, so
they provide no perfect safeguard.  As noted, fiddling with conditions
also may reduce error rates, but these will still be unacceptably high
for the average sized insert.  Besides, many mutations in cloned PCR
products reside in primer-encompassed sequences and are caused by primer
synthesis problems, not the PCR itself.  Finally, we and others have
observed that the mutation rate varies hugely between different template
sequences in a quite unpredictable way. 

ALWAYS sequence inserts that have been PCR cloned.

Best wishes,
-- 
Chris Boyd                      | from, but \    Medical Genetics Section
Chris.Boyd at ed.ac.uk             | not for,  /    MMC, Edinburgh Uni.,
http://www.ed.ac.uk/~cboyd                       EH4 2XU, SCOTLAND




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