Smolke:Protocols/Chromosomal Modification: Difference between revisions
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#Verify integration, either by [[Smolke:Protocols/Yeast Colony PCR|PCR]] or by functional tests. Realize that you might have integration, but at the wrong locus. | #Verify integration, either by [[Smolke:Protocols/Yeast Colony PCR|PCR]] or by functional tests. Realize that you might have integration, but at the wrong locus. | ||
#Select a strain with the correct integration. Grow in YPD for ~2 days, back diluting in the morning and evening. | #Select a strain with the correct integration. Grow in YPD for ~2 days, back diluting in the morning and evening. | ||
#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. | #Plate cells on [[Smolke:Protocols/5FOA|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. | ||
#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). | #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). | ||
#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). | #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). |
Revision as of 14:40, 20 October 2009
KnockoutsTalk to Mike. InsertionsPCR MethodTalk to Mike. Plasmid MethodOverviewWe use the disintegrator vectors[1] from EUROSCARF, stored as pCS1439 to 1441. Protocol
Notes
References |