nayers at utmem1.utmem.edu (nancy ayers) wrote:
>I have two questions: 1) What is the appropriate concentration of 3-AT on
>-HIS plates (I am using HF7c with a leaky promter and wish to reduce the #
>of false positive colonies I get). I have looked for it but am unable to
>locate this info. and 2) has anyone made a high expression vector with
>the full length ADH promoter for two-hybrid that has the same MCS as
>clonetech's original pGBT9 and pGAD424?? I am using these vectors now but
>would like to possibly move to higher expressing vectors...with relatively
>little effort. I know that they have vectors with the full length ADH but
>the MCS are different.
>Thanks!!! Any and all help is appreciated!
>nayers at utmem1.utmem.edu
We use strain Y190 from Clontech (which has leaky His expression) and
find that 25mM 3-AT final concentration in the -His plates works well for
screening. However, is your leaky expression in HF7c due to a change in
the yeast itself, or is it expression due to activation by your bait
plasmid?? When we were using HF7c we didn't use 3-AT at all.