help for site-directed mutagensis

Nagesh via methods%40net.bio.net (by nagesh.chakka from anu.edu.au)
Sun Feb 18 16:06:56 EST 2007


Hi Steven,
Try sequencing about 4 to 5 different colonies. I had a similar problem 
and when I went ahead sequencing more colonies from the same batch I 
could find the right colony with mutation (1 out of 7 sequenced was 
successful).
Good luck
Nagesh

>> Hi, Folks
>>
>> I have been doing site-directed mutagensis for  a while. My vector is
>> pcDNA 1.3 of ~5Kb plus 1.5 kb insert. I try to have point mutatation
>> in one site. Unfortunately, after sequencing, all i got are the
>> templates without any mutatation. I set up negative control, which has
>> no primers and been treated with Dpn1, and positive control without
>> being treated with Dpn1. Both controls works well, which means my
>> transformation works well.
>> I use 20 ng template in 25 ul reaction in PCR step. Is it too much for
>> 0.5 Dpn1 digestion?
>>
>> Any one give me suggestion??
>>
>> thanks
>>
>> steven
>>     
>
> Steven,
> Probably 20 ng is too much, but I will sugest something else - next
> time you make mutations to include silent mutation that create (or
> abolish) restriction site in your primer so you can screen your
> colonies before sequencing.
> my2c
> Peter
>
>
>   



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