Purify integrating PCR product before transforming yeast? - (Mar/06/2006 )
I'm trying to transform yeast of the strains W3031 MATa and W3031 MATalpha using the LiAc-method, aiming at getting a 2.8 kbp DNA construct to homologously recombine with one of the chromosomes. The construct is made in a PCR machine using the Expand system (Boehringer).
Transformation seems to fail. I can see faint reddish growth on all the plates after transformation, but no distinct colonies. I've tried to grow the "reddish growth" in selective medium, nothing grew. I've tried different concentrations of DNA.
Is it important to purify the PCR product before transformation? I've searched the literature, but it isn't mentioned anywhere.
Thank you for any suggestions!
What is your selection and do you need to pregrow without selection before you plate selection media.