Smolke:Protocols/Chromosomal Modification

Knockouts
Talk to Mike.

PCR Method
Talk to Isis.

Overview
We use the disintegrator vectors sadowski from EUROSCARF, stored as pCS1439 to 1441.

Protocol

 * 1) Clone your gene of interest into the appropriate integrating plasmid
 * 2) Prep the resulting plasmid and digest with an enzyme that only cuts the YIp-In. E.g. NruI for 1441, AleI for 1440.
 * 3) Transform your desired yeast strain using standard chemical methods. Select for growth on -URA.
 * 4) Patch colonies from the -URA plate onto a new -URA plate (both to save and to verify that they really are URA positive).
 * 5) Verify integration, either by PCR or by functional tests. Realize that you might have integration, but at the wrong locus.
 * 6) Select a strain with the correct integration. Grow in YPD for ~2 days, back diluting in the morning and evening.
 * 7) Plate cells on 5-FOA plates, being careful not to saturate the plate. If you have too many cells on the 5-FOA plate, restreak to single colonies on a new 5-FOA plate.
 * 8) Patch colonies from the 5-FOA plate onto a new 5-FOA plate (both to save, and to verify that they really are URA negative).
 * 9) Verify that the desired cassette is still integrated, either by PCR or by function. To further confirm, you can check that the cells cannot grow on the appropriate plates (-Lys/-Met/+5-FC).