making conditional mutants

[Kevin Morano]

FATIMA at AIMP.UNA.AC.AT wrote:
: 2. Give up the GAL promoter and try to make a thermosensitive allele
: by mutagenic PCR. It works quite fine - just reduce the concentration
: of one of the nucleotides by a factor of five and add MnCl2 (final
: conc. 0.5 mM) and mercaptoethanol (final conc. 10 mM) to the reaction.
: IMPORTANT: use a native Taq polymerase, not AmpliTaq - that wouldn't
: make any mutations.
>>>>>>

I have used this method essentially as Fatima describes it (sans BME) and 
it has produced loads os ts conditional alleles. I had to titrate the 
MnCL2 to get about %50 reduction in PCR yield based on added Mg. In fact, 
it really seems to be the Mn/Mg ratio that determines yield vs. 
poisoning. Haven't sequenced yet, so I don't know the extent of damage. 
The method is quite easy and efficient. Moreover, I used the "in vivo" 
ligation procedure: transforming ug quanitities of PCR product w/ 
homologous ends with ssDNA and lots of cut vector to have the yeast 
perform the sometimes difficult PCR cloning for me (template for PCR was 
the original gene cloned into parent vector). The mutagenesis took two 
days, from PCR to transformation. 

Good luck!
---------------------------------------------------------------------------
Kevin A. Morano
Internet:kamorano at ucdavis.edu    
Bitnet:kamorano at ucdavis   
Section of Microbiology, University of California, Davis