pcr products wont give me colonies - (Apr/11/2006 )
I have been carrying out PCR based site directed mutagenesis for past two weeks. I normaly run gel electrophoresis after PCR to see if i obtained a PCR product then i transform into xl1 blue supercompetent cells. I get the expected results from the gel but I dont understand why i dont get colonies after transforming. the cells are competent because i get loads of colonies from the PCR control sample and puc18. I have tried transforming with more PCR product ie 10x more. but still no colonies from samples. So what may be wrong.
is this quickchange or a two step PCR mutagenesis method?
it is the quick change.
I have also had this problem with a vector I have tried to site-direct. I must warn you that at the end I had to change my vector because none of the methods worked. Nonetheless, they are still worth a try.
The general guidelines of the site directed protocol I've used require amplification of the entire plasmid. I am assuming that after PCR you get rid of the template DNA using DpnI and clean your DNA on columns (in my lab we use Nucleospin, but any other columns are fine). AFTER these steps you should still be able to see a good amount plasmid DNA on a gel. Then you transform them into bacteria and in your case do not see any colonies.
Sometimes the plasmid is not very compatible with the bacteria you are using and they do not express it very well. Try using lower amouts of antibiotics (make sure you are using the correct antibiotic for your plasmid. It is a common mistake) to grow your transformed bacteria. If that doesn't work try using different types of competent bacteria (DH5alpha, XL1 blue etc.).
As I said, no guaranties, but I hope this will work.