Quickchange is obsolete :)
Artem
On Fri, Feb 3, 2012 at 7:56 PM, Roger Rowlett <[log in to unmask]> wrote:
> We prefer to do MEGAWHOP PCR and use 1-5 uL of the DPN digested PCR product.
> This is extremely reliable with commercial competent cells. See
> http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Recombinant+DNA+Protocols#Site_directed_mutagenesis_by_Megaprimer_Quick_Change_MEGAWHOP_PCR
> for details. Allow 1 min/KB each cycle for whole plasmid PCR extension.
>
> Roger Rowlett
>
> On Feb 3, 2012 8:29 PM, "Nian Huang" <[log in to unmask]> wrote:
>>
>> Just a reminder. Quickchange is not PCR. It is linear amplification. It is
>> very hard to see a band in the gel if you follow the standard protocol.
>>
>> Nian
>>
>> On Fri, Feb 3, 2012 at 12:14 PM, Fred <[log in to unmask]> wrote:
>>>
>>> Hi CCP4 list,
>>> Thanks everyone who have answered my post concerning to mutagenesis.
>>> From quick reading most of the answers, the following seems to be a
>>> consensus:
>>> 1) Do not concentrate your PCR product;
>>> 2) Too much DNA and/or impurities like salts or whatever can inhibits
>>> transformation;
>>> 3) Purify your PCR product before transformation if possible or use 3 of
>>> 4 microL of it. This is more or less the amount of DNA showed in the
>>> uploaded image.
>>> Kind regards,
>>> Fred
>>>
>>> P.S.: I'll let you know the results.
>>
>>
>
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