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Site-directed mutagenesis

Julie A. Kirihara kirihara at gold.tc.umn.edu
Mon Oct 14 10:44:24 EST 1996

wliu at receptor.pharm.uwa.edu.au wrote:

> How can assess the possibility of
>mutagenic oligo binding to template DNA  before I do the sequence
>analysis  if I don't set up a control reaction system containing only
>mutagenic oligos as a primer ( Promerga manual doesn't  suggest this
>type of control system)?
>Does anyone there has ideas about this ?


You can tell if the primer binds to your template by setting up a
sequencing reaction using your ssDNA as template and your mutagenic
primer as sequencing primer.  If you get a nice sequencing ladder,
then you can usually assume your primer will prime synthesis during
the mutagenesis reaction also.

Alternatively, and easier:

If at all possible, I try to include a restriction site change (new
site if possible, or removal of an existing site) into the design of a
mutagenic primer.  That way you can tell by restriction digests of
minipreps from your clones whether the mutagenic primer worked or not,
avoiding the need to do initial screening by sequencing.

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