Creating mutants (View forum version)



molecule

Posted 16 November 2009 - 03:11 PM

I have to create some double mutants to see how they function. I have created the single mutants in these combinations and cloned them to a plasmid. The size of the plasmid is ~11kb. In order to create the double mutants I would like to get some advice as to the best possible way to do it with minimal sequencing, and if possible eliminate the cloning process, (which I guess would not be easy!)

Stuck with Ligation

Posted 16 November 2009 - 03:56 PM

Simple; look up quickchange mutatgensis. Order 2 new primers for the mutation you want. Should take a day to run the PCR and transform and then an overnight to isolate enough DNA for sequencing. Very quick and very easy. No restriction enzymes or additional cloning steps or ligations needed.

Warren

Posted 16 November 2009 - 10:35 PM

I have to create some double mutants to see how they function. I have created the single mutants in these combinations and cloned them to a plasmid. The size of the plasmid is ~11kb. In order to create the double mutants I would like to get some advice as to the best possible way to do it with minimal sequencing, and if possible eliminate the cloning process, (which I guess would not be easy!)


Well, more details would be needed, but in an IDEAL situation, your single mutants would have a unique restriction site in between them, and one on one side or the other, and the double mutants could be created by simply switching fragments....it would require cloning obviously, but it would be extremely easy and no sequencing would be required (assuming your single mutants are already sequenced). However, more details are needed. Warren..

Vini

Posted 16 November 2009 - 10:56 PM

you can use the stratagene site directed mutagenesis kit....that way you can skip an extra cloning step