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yeast functional complementation assay


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#1 ajs

ajs

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Posted 18 September 2009 - 01:48 AM

Can anyone tell me a detailed protocol for functional complementation assay in yeast?

My strain of yeast is supposed to be deficient in hexose transporters (EBY.VW4000) and should not grow on any sugar except maltose. When i did a drop test using this strain (untransformed, just to see if the strain is all right), the yeast grew in glucose and mannose as well (where it should not grow). The drop test was done as follows:
I inoculated single colony from plate to 5 ml liquid culture (Yeast nitrogen base+aminoacids+ ammonium sulfate+maltose), grew that overnight. wash the cells two times in sterile water and then diluted the cell suspension to OD of 0.1 and 0.01 and spotted 10ul each on 3 different plates ccontaining the same basal media as above but with differnt sugars (maltose/ glucose/mannose). The yeast grows in all the three sugars (but it should not on glucose and mannose). what could be the problem?

does the use of a log phase suspension instead on O/N growth will change anything? or there is somethig wrong with the strain itself?

i have another query regarding media preperation. Can i autoclave YNB?

Thanks




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