Hi,
im using a stragene QikChange II site directed mutagenesis kit to do a site-directed mutagenesis study.
The plasmid im using is pGL3-basic. It is about 4.8kb + my insert 500bp approximately = 5.3kb.
So i did a PCR reaction at 95oC at 30secs.55oC at one minute and 68oC for 6 minute ( to play safe?) for 17 cycle.
No band was observed after the cycling when i run on gel.
But i continued with my Dpn I digestion as per protocol.
But i did not get any colonies. I tried using different concentration of plasmid 10ng-50ng ( nothing was observed.)
I used 1-2ul for my transformation - no colonies.
The cells im using is XL-1 competent cells ( from the kit itself)
Can anyone highlight to me which step i might have gone wrong? im a newbie to the kit.
Update:Thank you!
Answers & Comments
Verified answer
Site-directed mutagenesis is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule, usually a circular molecule known as a plasmid. In general, site-directed mutagenesis requires that the wild-type gene sequence be known.
This technique is also known as site-specific mutagenesis or Oligonucleotide-directed mutagenesis.
As for knowing how you may have run it better, one would have to retrace your steps. Mutation isn't something the average Joe knows about. My take is that you jumped ahead too early and didn't allow the band to be observed, or perhaps you "glanced" away right when it should have happened. I am purely speculating. Re-run from the beginning, after reading through entirely what the procedures are. Good luck.