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==Materials==
Per strain, this is enough for 10 transformations per strain
*50 mL autoclaved YPD in a 250 mL flask
*200 mL 2xYPD
*Autoclaved empty 250 mL flask
*Herring testes DNA (Clontech Cat# 17401336)
*50% PEG 3500, autoclaved
*1M Lithium Acetate, autoclaved
*Sterile water
==Protocol==
#Innoculate 50 mL YPD overnight in a 250 mL flask at 24C before going home
#The next morning check OD-600 of yeast.
##Add 10 uL cells to 1 mL of water and measure OD-600.
##Adjust for dilution factor (100X) and calculate OD.
##Dilute to a final OD of 0.5 in 50 mL of '''2xYPD''' in an empty autoclaved 250 mL flask.
#Incubate at 24C for 2 divisions (typically ~4.5h).
#Centrifuge cells at 3000g for 5min.
#Boil hering tested DNA for 5 min at 95C then cool on ice.
#Gently resuspend in 25mL sterile water.
#Centrifuge cells at 3000g for 5min.
#Gently resuspend cells in 1mL sterile water and transfer to an eppendorf tube
#Centrifige 30s in a microfuge and aspirate supernatant
#Resuspend with 1mL sterile water ensuring to break up any clumps.
#Transfer 100 uL aliquots of cells to 1.5mL tubes, one for each transformation.
#Prepare Transformation Mixture for the appropriate number of transformations:
{| border="1"
! || Reagent || Concentration || 1 || 5(6X) || 10(11X)
|-
|PEG 3500 || 50% || 240uL || 1440uL || 2640uL
|-
|Lithium Acetate || 1M || 36uL || 216uL || 396uL
|-
|Herring Testes DNA || 10mg/mL || 1uL || 60uL || 110uL
|-
|Water|| || 34uL || 204uL || 374uL
|-
|Tube || eppendorf tube || 15mL Falcon Tube || 15 mL Falcon Tube
|}
#Add 360 uL Transformation Mixture to each tube of cells and mix by vortexing.
#Incubate at 42C for 40min.
#Centrifuge for 30s and aspirate supernatant
#Add 1mL sterile water, resuspend and mix by gently vortexing.
#Plate 50 uL on appropriate selective plate.
#Allow to grow for 3-4 days at 24C.
Based on PMID 17401336. See http://home.cc.umanitoba.ca/~gietz/method.html
Per strain, this is enough for 10 transformations per strain
*50 mL autoclaved YPD in a 250 mL flask
*200 mL 2xYPD
*Autoclaved empty 250 mL flask
*Herring testes DNA (Clontech Cat# 17401336)
*50% PEG 3500, autoclaved
*1M Lithium Acetate, autoclaved
*Sterile water
==Protocol==
#Innoculate 50 mL YPD overnight in a 250 mL flask at 24C before going home
#The next morning check OD-600 of yeast.
##Add 10 uL cells to 1 mL of water and measure OD-600.
##Adjust for dilution factor (100X) and calculate OD.
##Dilute to a final OD of 0.5 in 50 mL of '''2xYPD''' in an empty autoclaved 250 mL flask.
#Incubate at 24C for 2 divisions (typically ~4.5h).
#Centrifuge cells at 3000g for 5min.
#Boil hering tested DNA for 5 min at 95C then cool on ice.
#Gently resuspend in 25mL sterile water.
#Centrifuge cells at 3000g for 5min.
#Gently resuspend cells in 1mL sterile water and transfer to an eppendorf tube
#Centrifige 30s in a microfuge and aspirate supernatant
#Resuspend with 1mL sterile water ensuring to break up any clumps.
#Transfer 100 uL aliquots of cells to 1.5mL tubes, one for each transformation.
#Prepare Transformation Mixture for the appropriate number of transformations:
{| border="1"
! || Reagent || Concentration || 1 || 5(6X) || 10(11X)
|-
|PEG 3500 || 50% || 240uL || 1440uL || 2640uL
|-
|Lithium Acetate || 1M || 36uL || 216uL || 396uL
|-
|Herring Testes DNA || 10mg/mL || 1uL || 60uL || 110uL
|-
|Water|| || 34uL || 204uL || 374uL
|-
|Tube || eppendorf tube || 15mL Falcon Tube || 15 mL Falcon Tube
|}
#Add 360 uL Transformation Mixture to each tube of cells and mix by vortexing.
#Incubate at 42C for 40min.
#Centrifuge for 30s and aspirate supernatant
#Add 1mL sterile water, resuspend and mix by gently vortexing.
#Plate 50 uL on appropriate selective plate.
#Allow to grow for 3-4 days at 24C.
Based on PMID 17401336. See http://home.cc.umanitoba.ca/~gietz/method.html
