Yeast Transformation - Revision history

Davebrid: typo with dna volume

2009-12-22T17:14:39Z

typo with dna volume

← Older revision		Revision as of 17:14, 22 December 2009
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	\|Lithium Acetate \|\| 1M \|\| 36uL \|\| 216uL \|\| 396uL		\|Lithium Acetate \|\| 1M \|\| 36uL \|\| 216uL \|\| 396uL
	\|-		\|-
	\|Herring Testes DNA \|\| 10mg/mL \|\| ~~1uL~~ \|\| 60uL \|\| 110uL		\|Herring Testes DNA \|\| 10mg/mL \|\| 10uL \|\| 60uL \|\| 110uL
	\|-		\|-
	\|Water\|\| \|\| 34uL \|\| 204uL \|\| 374uL		\|Water\|\| \|\| 34uL \|\| 204uL \|\| 374uL

Davebrid: table formatting error

2009-12-22T16:48:09Z

table formatting error

← Older revision		Revision as of 16:48, 22 December 2009
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	#Prepare Transformation Mixture for the appropriate number of transformations:		#Prepare Transformation Mixture for the appropriate number of transformations:
	{\| border="1"		{\| border="1"
	! \|\| Reagent \|\| Concentration \|\| 1 \|\| 5(6X) \|\| 10(11X)		! Reagent \|\| Concentration \|\| 1 \|\| 5(6X) \|\| 10(11X)
	\|-		\|-
	\|PEG 3500 \|\| 50% \|\| 240uL \|\| 1440uL \|\| 2640uL		\|PEG 3500 \|\| 50% \|\| 240uL \|\| 1440uL \|\| 2640uL
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	\|Water\|\| \|\| 34uL \|\| 204uL \|\| 374uL		\|Water\|\| \|\| 34uL \|\| 204uL \|\| 374uL
	\|-		\|-
	\|Tube \|\| eppendorf tube \|\| 15mL Falcon Tube \|\| 15 mL Falcon Tube		\|Tube \|\| \|\| eppendorf tube \|\| 15mL Falcon Tube \|\| 15 mL Falcon Tube
	\|}		\|}

Davebrid at 17:56, 21 December 2009

2009-12-21T17:56:34Z

← Older revision		Revision as of 17:56, 21 December 2009
Line 8:		Line 8:
	*1M Lithium Acetate, autoclaved		*1M Lithium Acetate, autoclaved
	*Sterile water		*Sterile water
	*Selective Agar Plates (see [[Yeast Selective Media and Agar]]		*Selective Agar Plates (see [[Yeast Selective Media and Agar]])

	==Protocol==		==Protocol==

Davebrid: added link to agar plates

2009-12-21T17:56:25Z

added link to agar plates

← Older revision		Revision as of 17:56, 21 December 2009
Line 8:		Line 8:
	*1M Lithium Acetate, autoclaved		*1M Lithium Acetate, autoclaved
	*Sterile water		*Sterile water
			*Selective Agar Plates (see [[Yeast Selective Media and Agar]]

	==Protocol==		==Protocol==

Davebrid: wrote initial page

2009-12-21T17:54:27Z

wrote initial page

New page

==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