PCR site-directed mutagenesis

D.K. dk at no.email.thankstospam.net
Fri Jul 19 08:07:08 EST 2002


 Philipp Wechner <philipp.wechner at uibk.ac.at> wrote:

>
>what do you mean with PCR1 and 2?

Sounds like it was overlap extension PCR. 

>amarindc at aol.com schrieb:
>
>> We have been trying to do mutagenesis in certain area of a protein. Three
> people in my lab are using different set of mutant primers for different aa.
> mutations. The first PCR goes fine, so apparently does the second. But we are
> unable to either clone or reamplify the insert using the outside flanking
> primers (used in PCR 2). We have already tried different conc. of DNA,
> primers, purification, annealing temp., number of cycles... We have no idea
> whatsoever on what is going on.
>>
>> By the way: we have already mutated other aa. in this same region and worked
> out fine!
>>
>> Thanx,
>>
>> Ana
>>
>>
> http://biowww.net/mynews/tree.php?group_name=bionet_molbio_methds-reagnts&begi
>n=0
>
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><!doctype html public "-//w3c//dtd html 4.0 transitional//en">
><html>
>what do you mean with PCR1 and 2?
><p>amarindc at aol.com schrieb:
><blockquote TYPE=CITE>We have been trying to do mutagenesis in certain
>area of a protein. Three people in my lab are using different set of mutant
>primers for different aa. mutations. The first PCR goes fine, so apparently
>does the second. But we are unable to either clone or reamplify the insert
>using the outside flanking primers (used in PCR 2). We have already tried
>different conc. of DNA, primers, purification, annealing temp., number
>of cycles... We have no idea whatsoever on what is going on.
><p>By the way: we have already mutated other aa. in this same region and
>worked out fine!
><p>Thanx,
><p>Ana
><p><a
> href="http://biowww.net/mynews/tree.php?group_name=bionet_molbio_methds-reagnt
>s&begin=0">http://biowww.net/mynews/tree.php?group_name=bionet_molbio_methds-re
>agnts&amp;begin=0</a></blockquote>
></html>
>
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