High Efficiency Transformation - Revision history

Torsten Waldminghaus at 17:25, 23 February 2009

2009-02-23T17:25:05Z

←Older revision		Revision as of 17:25, 23 February 2009
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		+	{{back to protocols}}
	Day 0		Day 0
	*Make sure you have the necessary solutions (instructions for how to make them can be found [http://www.umanitoba.ca/faculties/medicine/units/biochem/gietz/Solutions.html here]):		*Make sure you have the necessary solutions (instructions for how to make them can be found [http://www.umanitoba.ca/faculties/medicine/units/biochem/gietz/Solutions.html here]):

Tk at 04:11, 10 February 2008

2008-02-10T04:11:14Z

←Older revision		Revision as of 04:11, 10 February 2008
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	Notes:		Notes:
	*Based on Gietz et al. http://www.umanitoba.ca/faculties/medicine/units/biochem/gietz/method.html		*Based on Gietz et al. http://www.umanitoba.ca/faculties/medicine/units/biochem/gietz/method.html
		+	*See also the Gietz lab notes http://home.cc.umanitoba.ca/~gietz/
	*This protocol has been trimmed for conciseness. The original has some differences and some notes on how to achieve higher efficiency		*This protocol has been trimmed for conciseness. The original has some differences and some notes on how to achieve higher efficiency

Reshma P. Shetty at 21:30, 8 May 2007

2007-05-08T21:30:31Z

←Older revision		Revision as of 21:30, 8 May 2007
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	*Based on Gietz et al. http://www.umanitoba.ca/faculties/medicine/units/biochem/gietz/method.html		*Based on Gietz et al. http://www.umanitoba.ca/faculties/medicine/units/biochem/gietz/method.html
	*This protocol has been trimmed for conciseness. The original has some differences and some notes on how to achieve higher efficiency		*This protocol has been trimmed for conciseness. The original has some differences and some notes on how to achieve higher efficiency
		+
		+	[[Category:Protocol]]
		+	[[Category:In vivo]]
		+	[[Category:Yeast]]

Freimoser at 23:35, 20 October 2006

2006-10-20T23:35:36Z

←Older revision		Revision as of 23:35, 20 October 2006
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	Notes:		Notes:
-	*Based on ~~Geitz~~ et al. http://www.umanitoba.ca/faculties/medicine/units/biochem/gietz/method.html	+	*Based on Gietz et al. http://www.umanitoba.ca/faculties/medicine/units/biochem/gietz/method.html
	*This protocol has been trimmed for conciseness. The original has some differences and some notes on how to achieve higher efficiency		*This protocol has been trimmed for conciseness. The original has some differences and some notes on how to achieve higher efficiency

Amallet at 21:11, 31 March 2006

2006-03-31T21:11:07Z

←Older revision		Revision as of 21:11, 31 March 2006
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	#Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells.		#Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells.
	#* If your cell titer was higher/lower than 2x10<sup>7</sup> cells/ml, add proportionally more/less water at this step. Also, note that if you split your initial 50ml of culture into multiple tubes, the water should be split appropriately between the tubes.		#* If your cell titer was higher/lower than 2x10<sup>7</sup> cells/ml, add proportionally more/less water at this step. Also, note that if you split your initial 50ml of culture into multiple tubes, the water should be split appropriately between the tubes.
-	#Pipette 100 µl samples (ca. 10<sup>8</sup> cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at ~~top speed~~ for 30 sec and remove the supernatant.	+	#Pipette 100 µl samples (ca. 10<sup>8</sup> cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at 3000g for 30 sec and remove the supernatant.
	#Make up sufficient Transformation Mix for the planned number of transformations plus one extra. Keep the Transformation Mix in ice/water.		#Make up sufficient Transformation Mix for the planned number of transformations plus one extra. Keep the Transformation Mix in ice/water.
	:It's generally easiest to make a master mix of PEG, LiAc and the SS-carrier DNA and add 326 µl from the master mix to each transformation tube before adding the plasmid DNA + water component.		:It's generally easiest to make a master mix of PEG, LiAc and the SS-carrier DNA and add 326 µl from the master mix to each transformation tube before adding the plasmid DNA + water component.

Amallet at 21:10, 31 March 2006

2006-03-31T21:10:32Z

←Older revision		Revision as of 21:10, 31 March 2006
Line 18:		Line 18:
	#* If the cell titer is higher/lower than 2x10<sup>7</sup> cells/ml, you'll dilute it back down/up in a subsequent step		#* If the cell titer is higher/lower than 2x10<sup>7</sup> cells/ml, you'll dilute it back down/up in a subsequent step
	#* Now would also be a good time to start warming up the 42°C water bath you'll need later on		#* Now would also be a good time to start warming up the 42°C water bath you'll need later on
-	#Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.	+	#Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge at 3000 g for 30 sec and discard the supernatant.
	#Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells.		#Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells.
	#* If your cell titer was higher/lower than 2x10<sup>7</sup> cells/ml, add proportionally more/less water at this step. Also, note that if you split your initial 50ml of culture into multiple tubes, the water should be split appropriately between the tubes.		#* If your cell titer was higher/lower than 2x10<sup>7</sup> cells/ml, add proportionally more/less water at this step. Also, note that if you split your initial 50ml of culture into multiple tubes, the water should be split appropriately between the tubes.
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	#Add 360 µl of Transformation Mix to each transformation tube and resuspend the cells by vortex mixing vigorously.		#Add 360 µl of Transformation Mix to each transformation tube and resuspend the cells by vortex mixing vigorously.
	#Incubate the tubes in a 42°C water bath for 40 min.		#Incubate the tubes in a 42°C water bath for 40 min.
-	#Microcentrifuge at ~~top speed~~ for 30 sec and remove the Transformation Mix with a micropipettor.	+	#Microcentrifuge at 3000 g for 30 sec and remove the Transformation Mix with a micropipettor.
	#Pipette 1.0 ml of sterile water into each tube; stir the pellet by with a micropipette tip and vortex .		#Pipette 1.0 ml of sterile water into each tube; stir the pellet by with a micropipette tip and vortex .
	#*We like to be a gentle as possible at this step if high efficiency is important. Excessive washing washes away transformants.		#*We like to be a gentle as possible at this step if high efficiency is important. Excessive washing washes away transformants.

Amallet at 23:23, 9 March 2006

2006-03-09T23:23:20Z

←Older revision		Revision as of 23:23, 9 March 2006
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	#Pipette 100 µl samples (ca. 10<sup>8</sup> cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at top speed for 30 sec and remove the supernatant.		#Pipette 100 µl samples (ca. 10<sup>8</sup> cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at top speed for 30 sec and remove the supernatant.
	#Make up sufficient Transformation Mix for the planned number of transformations plus one extra. Keep the Transformation Mix in ice/water.		#Make up sufficient Transformation Mix for the planned number of transformations plus one extra. Keep the Transformation Mix in ice/water.
-	:It's generally easiest to make a master mix of PEG, LiAc and the SS-carrier DNA and add ~~324~~ µl from the master mix to each transformation tube before adding the plasmid DNA + water component.	+	:It's generally easiest to make a master mix of PEG, LiAc and the SS-carrier DNA and add 326 µl from the master mix to each transformation tube before adding the plasmid DNA + water component.
	:For the plasmid DNA + water component, you'll need to calculate, for each transformation, what vol. of your plasmid DNA solution contain the appropriate amount of plasmid DNA (0.5-1.0 µg). Add in the appropriate amount of plasmid DNA solution and then make it up to 34µl with water.		:For the plasmid DNA + water component, you'll need to calculate, for each transformation, what vol. of your plasmid DNA solution contain the appropriate amount of plasmid DNA (0.5-1.0 µg). Add in the appropriate amount of plasmid DNA solution and then make it up to 34µl with water.
	::{\| border="1" cellpadding="2" cellspacing="0"		::{\| border="1" cellpadding="2" cellspacing="0"

Amallet at 13:51, 30 November 2005

2005-11-30T13:51:07Z

←Older revision		Revision as of 13:51, 30 November 2005
Line 1:		Line 1:
		+	Day 0
		+	*Make sure you have the necessary solutions (instructions for how to make them can be found [http://www.umanitoba.ca/faculties/medicine/units/biochem/gietz/Solutions.html here]):
		+	*# Single-stranded carrier DNA
		+	*# PEG 3350 50% w/vol
		+	*# 1.0 M LiAc
		+	*Make sure you have enough plasmid DNA for your transformations. Each transformation should use between 0.5 and 1.0 µg of plasmid.
		+
	Day 1		Day 1
	Inoculate the yeast strain into 5 ml of liquid medium and incubate overnight.		Inoculate the yeast strain into 5 ml of liquid medium and incubate overnight.
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	#Transfer 50 ml of YPD to the pre-warmed culture flask and add 2.5 x 10<sup>8</sup> cells to give 5 x 10<sup>6</sup> cells/ml.		#Transfer 50 ml of YPD to the pre-warmed culture flask and add 2.5 x 10<sup>8</sup> cells to give 5 x 10<sup>6</sup> cells/ml.
	#Incubate the flask on a rotary or reciprocating shaker at 30°C and 200 rpm.		#Incubate the flask on a rotary or reciprocating shaker at 30°C and 200 rpm.
-	#Boil a 1.0 ml sample of carrier DNA for 5 min and chill in an ice/water bath while harvesting the cells.	+	#Boil a 1.0 ml sample of carrier DNA for 5 min and chill in an ice/water bath while harvesting the cells (the next step).
	#*It is not necessary or desirable to boil the carrier DNA every time. Keep a small aliquot in your own freezer box and boil after 3-4 freeze-thaws. But keep on ice when out.		#*It is not necessary or desirable to boil the carrier DNA every time. Keep a small aliquot in your own freezer box and boil after 3-4 freeze-thaws. But keep on ice when out.
	#When the cell titer is at least 2 x 10<sup>7</sup> cells/ml, which should take about 4 hours, harvest the cells by centrifugation at 3000 g for 5 min, wash the cells in 25 ml of sterile water and resuspend in 1 ml of sterile water.		#When the cell titer is at least 2 x 10<sup>7</sup> cells/ml, which should take about 4 hours, harvest the cells by centrifugation at 3000 g for 5 min, wash the cells in 25 ml of sterile water and resuspend in 1 ml of sterile water.
		+	#* If the cell titer is higher/lower than 2x10<sup>7</sup> cells/ml, you'll dilute it back down/up in a subsequent step
		+	#* Now would also be a good time to start warming up the 42°C water bath you'll need later on
	#Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.		#Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.
	#Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells.		#Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells.
		+	#* If your cell titer was higher/lower than 2x10<sup>7</sup> cells/ml, add proportionally more/less water at this step. Also, note that if you split your initial 50ml of culture into multiple tubes, the water should be split appropriately between the tubes.
	#Pipette 100 µl samples (ca. 10<sup>8</sup> cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at top speed for 30 sec and remove the supernatant.		#Pipette 100 µl samples (ca. 10<sup>8</sup> cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at top speed for 30 sec and remove the supernatant.
-	#Make up sufficient Transformation Mix for the planned number of transformations plus one extra. Keep the Transformation Mix in ice/water.	+	#Make up sufficient Transformation Mix for the planned number of transformations plus one extra. Keep the Transformation Mix in ice/water.
		+	:It's generally easiest to make a master mix of PEG, LiAc and the SS-carrier DNA and add 324 µl from the master mix to each transformation tube before adding the plasmid DNA + water component.
		+	:For the plasmid DNA + water component, you'll need to calculate, for each transformation, what vol. of your plasmid DNA solution contain the appropriate amount of plasmid DNA (0.5-1.0 µg). Add in the appropriate amount of plasmid DNA solution and then make it up to 34µl with water.
	::{\| border="1" cellpadding="2" cellspacing="0"		::{\| border="1" cellpadding="2" cellspacing="0"
	\|-		\|-
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	!Reagents\|\|1\|\|5 (6X)\|\|10 (11X)		!Reagents\|\|1\|\|5 (6X)\|\|10 (11X)
	\|-		\|-
-	\|PEG ~~3500~~ 50% w/v\|\|240 µl\|\|1440 µl\|\|2640 µl	+	\|PEG 3350 50% w/v\|\|240 µl\|\|1440 µl\|\|2640 µl
	\|-		\|-
	\|LiAc 1.0 M\|\|36 µl\|\|216 µl\|\|396 µl		\|LiAc 1.0 M\|\|36 µl\|\|216 µl\|\|396 µl
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	#Pipette 1.0 ml of sterile water into each tube; stir the pellet by with a micropipette tip and vortex .		#Pipette 1.0 ml of sterile water into each tube; stir the pellet by with a micropipette tip and vortex .
	#*We like to be a gentle as possible at this step if high efficiency is important. Excessive washing washes away transformants.		#*We like to be a gentle as possible at this step if high efficiency is important. Excessive washing washes away transformants.
-	#Plate appropriate dilutions of the cell suspension onto SC selection medium. For transformation with an integrating plasmid (YIp), linear construct or oligonucleotide, plate 200 µl onto each of 5 plates; for a YEp, YRp or YCp library plasmid dilute 10 µl of the suspension into 1.0 µl of water and plate 10 and 100 µl samples onto two plates each. The 10 µl samples should be pipetted directly into 100 µl puddles of sterile water on the SC selection medium.	+	#Plate appropriate dilutions of the cell suspension onto SC selection medium. For transformation with an integrating plasmid (YIp), linear construct or oligonucleotide, plate 200 µl onto each of 5 plates; for a YEp, YRp or YCp library plasmid dilute 10 µl of the suspension into 1.0 ml of water and plate 10 and 100 µl samples of the dilute onto two plates each. The 10 µl samples should be pipetted directly into 100 µl puddles of sterile water on the SC selection medium.

	Notes:		Notes:
	*Based on Geitz et al. http://www.umanitoba.ca/faculties/medicine/units/biochem/gietz/method.html		*Based on Geitz et al. http://www.umanitoba.ca/faculties/medicine/units/biochem/gietz/method.html
	*This protocol has been trimmed for conciseness. The original has some differences and some notes on how to achieve higher efficiency		*This protocol has been trimmed for conciseness. The original has some differences and some notes on how to achieve higher efficiency

Jgritton at 20:33, 8 June 2005

2005-06-08T20:33:42Z

←Older revision		Revision as of 20:33, 8 June 2005
Line 3:		Line 3:

	Day 2		Day 2
-	#Determine the titer of the yeast culture by pipetting 10 µl of cells into 1.0 ml of water in a spectrophotometer cuvette and measuring the OD at 600 nm. For many yeast strains a suspension containing 1 x 10^6 cells/ml will give an ~~OD600~~ of 0.1.	+	#Determine the titer of the yeast culture by pipetting 10 µl of cells into 1.0 ml of water in a spectrophotometer cuvette and measuring the OD at 600 nm. For many yeast strains a suspension containing 1 x 10<sup>6</sup> cells/ml will give an OD<sub>600</sub> of 0.1.
-	#Transfer 50 ml of YPD to the pre-warmed culture flask and add 2.5 x 10^8 cells to give 5 x 10^6 cells/ml.	+	#Transfer 50 ml of YPD to the pre-warmed culture flask and add 2.5 x 10<sup>8</sup> cells to give 5 x 10<sup>6</sup> cells/ml.
	#Incubate the flask on a rotary or reciprocating shaker at 30°C and 200 rpm.		#Incubate the flask on a rotary or reciprocating shaker at 30°C and 200 rpm.
	#Boil a 1.0 ml sample of carrier DNA for 5 min and chill in an ice/water bath while harvesting the cells.		#Boil a 1.0 ml sample of carrier DNA for 5 min and chill in an ice/water bath while harvesting the cells.
	#*It is not necessary or desirable to boil the carrier DNA every time. Keep a small aliquot in your own freezer box and boil after 3-4 freeze-thaws. But keep on ice when out.		#*It is not necessary or desirable to boil the carrier DNA every time. Keep a small aliquot in your own freezer box and boil after 3-4 freeze-thaws. But keep on ice when out.
-	#When the cell titer is at least 2 x ~~107~~ cells/ml, which should take about 4 hours, harvest the cells by centrifugation at 3000 g for 5 min, wash the cells in 25 ml of sterile water and resuspend in 1 ml of sterile water.	+	#When the cell titer is at least 2 x 10<sup>7</sup> cells/ml, which should take about 4 hours, harvest the cells by centrifugation at 3000 g for 5 min, wash the cells in 25 ml of sterile water and resuspend in 1 ml of sterile water.
	#Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.		#Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.
	#Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells.		#Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells.
-	#Pipette 100 µl samples (ca. ~~108~~ cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at top speed for 30 sec and remove the supernatant.	+	#Pipette 100 µl samples (ca. 10<sup>8</sup> cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at top speed for 30 sec and remove the supernatant.
	#Make up sufficient Transformation Mix for the planned number of transformations plus one extra. Keep the Transformation Mix in ice/water.		#Make up sufficient Transformation Mix for the planned number of transformations plus one extra. Keep the Transformation Mix in ice/water.
	::{\| border="1" cellpadding="2" cellspacing="0"		::{\| border="1" cellpadding="2" cellspacing="0"

Jgritton at 18:27, 24 May 2005

2005-05-24T18:27:05Z

←Older revision		Revision as of 18:27, 24 May 2005
Line 39:		Line 39:
	Notes:		Notes:
	*Based on Geitz et al. http://www.umanitoba.ca/faculties/medicine/units/biochem/gietz/method.html		*Based on Geitz et al. http://www.umanitoba.ca/faculties/medicine/units/biochem/gietz/method.html
-	*This protocol has been trimmed for ~~efficiency~~. The original has some differences and some notes on how to achieve higher efficiency	+	*This protocol has been trimmed for conciseness. The original has some differences and some notes on how to achieve higher efficiency