problem with site mutation - (Mar/06/2008 )

Hi, Dear all:
Now i have a problem with site muation. After liagation, i do a miniprep and sequence the N-terminal of the liagated plasmid to see if tag and and enzyme site is there. then i did a site mutation , however i did not see any colony even in the control plasmid.
can any1 give some suggestions? i have worked on it for a long time and i really get out of it.
thanks.
Niu

i used pfs DNA polymerase to do the pcr. re site is 5' BglII and 3' SacII. 5' has flag tag.
I used pEGFP-N1 vector, which is also re digested with the same enzyme.
after PCR, I purify the product and R.E PCR product, then i ligate the PCR product with vector. i have a lot of clones. i did a enyzme digest to confirm then i send 2 samples for sequnces of the 5'. then i did the site mutatin using stratagen kit and transform the competent cells. then i found no colonies grow, even the positive control ( control plasmid comes with the kit).
what is your suggestions? please help.
Niu

stratagene has a troubleshooting guide in the back of their protocol manual. If you're getting absolutely no colonies with the control, then I would recommend to check your plates first, then go through their checklist. There are really only a few variables that can go wrong with the control so it should be pretty easy to figure out.

check out cell competency and also donot over digest with DpnI.

Get new dNTP's and go over the mixture carefully.

No colonies sounds like the transformation is not working. I'm not sure what the positive control is in this procedure but transform some plain vector DNA into the cells to make sure they are competent and your transformation procedure is working. Then you can work out whether it is the Quickchange method that is failing. If the Quickchange doesn't work for you, you can always mutate the sequence by PCRing the insert with the sequence you want and cloning that into the vector. It may take a little longer but it's another good option.