Site-directed mutagenesis troubleshooting

Wolfgang Schechinger hubahopp at gmx.de
Mon May 10 04:40:38 EST 2004


Dear ?

increase the number of cycles to 25 and extend for up to 10 min. if this
does not work, decrease the annealing temp. when you run a sample (before
the DpnI) in agarose, can you see anything?

Wo 


At 07:54 10.05.2004 -0000, stoychevs at biology.biol.wits.ac.za wrote:
>I am attempting to create a truncated mutant by introducing a stop codon
into my primers using Stratagene's Quickchange Site-directed mutagenesis
kit. I am experiencing problems with my PCR since after transformation I
don't get any colonies on the plates (100mg/ml Amp). I know that the
transformaiton works since I transformed the wild-type plasmid
successesfully and got 100s of colonies. I have tried varying my template
DNA concentration (0.7ng - 100ng) while keeping the primer concentration in
excess (600ng) with no success. My plasmid (pGEX-4t-1) together with the
insert is 5.6kb. I use the following conditions for the PCR: 
>Segment 1: 1 cycle at 95C for 30sec 
>Segment 2: 16 cycles 95C for 30sec 
>55C for 1min 
>68C for 6min 
>Could you please advise on where I might be going wrong. Thank you.
>
>
>http://biowww.net/mynews/tree.php?group_name=bionet_molbio_methds-reagnts&b
egin=0
>
>
Wolfgang Schechinger


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