Genetics Laboratory BL415, Spring 2015 Yeast Transformation

Outline Yeast Transformation Set-up (March 24 th ) Yeast Replica-plating (March 31 st ) Yeast Screening for LEU2 and HIS3 genes (April 7 th )

The ‘wild-type’ strain YJJ662 that we will use has three different mutations and requires three nutrients: uracil (ura3 mutation) leucine (leu2 mutation) histidine (his3 mutation) We will transform this ‘wild-type’ strain with a shuttle vector containing the URA3 gene, and will select for colonies able to grow on media lacking uracil (SC-Ura, synthetic complete media lacking uracil). Then we will test which of the URA3 colonies have acquired a plasmid containing a particular DNA insert from the totality of the yeast genome library, namely we will ask which colonies acquired the LEU2 or the HIS3 gene, by replica- plating colonies from the SC-Ura plates onto plates lacking leucine (SC-Ura-Leu) or histidine (SC-Ura-His). We expect many yeast cells will be transformed with the plasmid vector and will become URA3 (the gene used for selection), but that only a few will get either the HIS3 or LEU2 gene (1/1500).

Transformation a)Transform 2 tubes of yeast using the lithium acetate (LiOAc) procedure : one gets no DNA (- control) and one gets 1  g of yeast library DNA. b)Plate each set of the transformants on 2 SC-Ura plates (synthetic complete media lacking uracil); incubate at 30 o C for 2-6 days (normal temperature for yeast). c)Plate dilutions of one of the yeast cultures on nonselective (YEPD) media to determine the total number of yeast in the culture. d)Plate a small amount of one of the yeast cultures on the YEPD media to verify that the yeast were alive after the chemical treatments.

Replica-plating a)Count the numbers of yeast cells on the YEPD plates to determine initial cell count: only count plates with about 30 to 300 colonies; estimate counts for crowded plates by counting sector. Remember that dilutions were plated, so calculate the number of cells/0.1 ml plated if the culture had not been diluted. b)Estimate the numbers of transformants on SC-Ura plates: count the colonies if <300; count a sector (1/4 or 1/8 of plate) if lots of colonies. Score TNTC (too numerous to count) if very crowded. c)Calculate the transformation frequency = the number of cells per 0.1 ml plated on SC-Ura plate divided by the number of undiluted cells/0.1 ml determined from the YEPD plates.This number is probably about (i.e. about 1/100,000 cells picks up the plasmid). d)Test if any URA3 transformants also got the LEU2 or HIS 3 genes from the library: replica-plate the colonies onto SC-Ura-Leu and SC-Ura-His media (plus SC-Ura media). Use sterile velveteen patches and the replica plate blocks. * if you only have a few colonies, use sterile toothpicks to transfer them to new media.

Screening for LEU2 and HIS3 genes Should one plasmid from the library be able to confer both LEU2 and HIS3? a)Count the numbers of colonies on the different media to determine if any transformants acquired the LEU2 or HIS3 genes.*** Save any colonies that did get these genes; I will later extract their plasmid DNA and analyze the inserts for size and specific other gene sequences carried. b)Calculate for your data: i) transformation frequency, ii) selection of a particular gene from the library. c)Obtain class data for: i) transformation frequency; ii) selection of particular genes from library. Be certain you understand how these data are calculated.