YG 2015 Week 1
Monday
Project 1
Screen for metabolic
mutation
(inoculate cells on
Sat/Sun? in YPD
BY4741/BY4742)
Project 2
Mating, Sporulation and
Tetrad Analysis,
Linkage studies
Tuesday (1) 31.3
1.EMS mutagenesis
2.UV mutagenesis
Plate cells on FAA
Mating of #23 to tester
strains
Mating of W1536 strains
to BY4741
Mating of respiratory
deficient mutants
Project 3
Sorting of mutants into
complementation groups
Project 4
Plasmid shuffle and
carbon source
effect
(inoculate
Project 5: Gene
Replacement
(template DNA)
pTSV30 ELO3-H2)
Project 6 Preparation of (Assistants: about
sequencing needs on
Library DNA and
week 4!!!!!)
cloning of mutations
Transform pTSV30
ELO3-H2 cells with
RFT1 plasmid
Wednesday (2) 1.4
Thursday
Friday
-----
Plating of mated cells
– selection for
Diploids
Plating of mated
cells – selection for
diploids
-------
PCR replacement
cassette
Cleanup of PCR product
Transform/plate E. coli cells
(yeast genomic DNA
libraries)
Large Scale DNA prep
(Assistants: move
plates to the fridge!)
(Assistants: move
plates to the fridge!)
YG 2015 Week 2
Monday
Score plates
Pick and re-plate
mutants (YPD)
Project 1
Screen for metabolic
mutation
Project 2
Mating, Sporulation
and Tetrad Analysis
Project 6
Preparation of Lib.
DNA and cloning of
mutations
Wednesday (4) 8.4.
Test mutants
On FAA/SC-Trp
Sporulation
Project 3
Sorting of mutants
into complementation
groups
Project 4
Plasmid shuffle
and carbon source
effect
Project 5
Gene Replacement
Tuesday (3) 7.4.
Thursday
Friday (5) 10.4.
Verify mutant phenotype
Mate mutants
Sporulation, first
tetrad dissections
Streak diploids on
YPD/SC-glycerol
Pick up single
colonies,
Streak for single
colonies
(Course assistants
inoculate 10 ml
cultures in the
morning!)
Pick whites and red
control colonies
Pick candidates
Test for geneticin
resistance
Transformation
Digest, run out on gel and
evaluate
Inoculate strain(s)
Library transformation
Pick colonies/restreak
YG 2015 Week 3
Monday 13.4
Selection of diploids
Project 1
Screen for metabolic
mutation
Project 2
Mating, Sporulation
and Tetrad Analysis,
Linkage studies
Score mutants
Inoculate selected mutants
Dissections
Project 3
Sorting of mutants into
complementation groups
Project 4
Plasmid shuffle
and carbon source
effect
Project 5: Gene
Replacement
Project 6
Preparation of Lib.
DNA and cloning of
mutations
Tetrad Dissection
Tuesday (6) 14.4
Dissections,
Replica plating
Score
-----
Pick whites and
control colonies?
Inoculate
Spotting assay
Pick up colonies
Restreak
Inoculate into SCD-Ura
Thursday 16.4
Friday 17.4
Transform selected mutants
with TRP1 plasmid
Dissections
Replica plating
Inoculate Geneticin
resistant strains
(Assistants: move
plates to fridge)
Wednesday (7) 15.4
Dissections
Dissections
(Assistants check
plates and
transfer to fridge
when grown far
enough)
Isolate genomicDNA
(need glass beads!)
Isolate yeast
genomic/plasmid DNA
Transform into E. coli
(Assistants move
transformation plates to
+4oC fridge)
Every day is a good day for tetrad dissections! (2-3 students/day)
YG 2015 Week 4
Monday
Project 1
Screen for metabolic
mutation
Project 2
Mating, Sporulation
and Tetrad Analysis
Project 3
Sorting of mutants into
complementation groups
Project 4
Plasmid shuffle
and carbon source
effect
Project 5
Gene Replacement
Project 6
Preparation of
Library DNA and
metabolic mutation
cloning
Tuesday (8) 21.4.
Thursday
Friday (10) 24.4.
Complementation analysis of
Diploids on –trp/5FAA; Score
TRP1 transformants
Take pictures of plates
Score complementation
analysis/Review of results
Replica plating
Take pictures of plates
Score
Review of results
------
Take pictures of plates
Review of results
Score
Pictures of Plates
Review of results
PCR analysis of knockouts
assistants
(inoculate E. coli
transformants)
Wednesday (9) 22.4.
First thing in the morning:
E. coli plasmid DNA preps
Prepare DNA and primers for
sequencing services (before
13:00)
Run out PCR to
confirm
Inoculate cells for
stocks? Pictures
Pictures
Make stocks?
Review of results
Blasting search of
sequences
Review of results