Lidstrom:Competent Cell Preparation - Revision history

Janet B. Matsen: /* Electrocompetent Cells */

Janet B. Matsen — Sun, 31 Mar 2013 15:17:59 GMT

Electrocompetent Cells

←Older revision		Revision as of 15:17, 31 March 2013
Line 163:		Line 163:
	## If using the tube spec, the path length is longer than in the cuvettes. Divide tube specs by 1.65 to convert to the 1 cm path length OD. (If using tube spec, let the OD get to ~1.)		## If using the tube spec, the path length is longer than in the cuvettes. Divide tube specs by 1.65 to convert to the 1 cm path length OD. (If using tube spec, let the OD get to ~1.)
	#Resuspend the pellet in a small volume, ideally up to 1/500<sup>ths</sup> of the culture volume.		#Resuspend the pellet in a small volume, ideally up to 1/500<sup>ths</sup> of the culture volume.
-	## See ~~graph below.~~ [[User:Janet B. Matsen\|Janet]] ~~did this experiment~~. The really concentrated cells performed well despite having clumped into a serious "booger" during recovery (pre-plating) that was mostly unspreadable. [[image:2012_11 electroporation - number of colonies versus competent cell density.jpg\|thumb\|center\|electroporation: number of colonies versus competent cell density]]	+	## See [[User:Janet B. Matsen\|Janet's]] graph below. The really concentrated cells performed well despite having clumped into a serious "booger" during recovery (pre-plating) that was mostly unspreadable. [[image:2012_11 electroporation - number of colonies versus competent cell density.jpg\|thumb\|center\|electroporation: number of colonies versus competent cell density]]
-	#Aliquot into 1.5 ml centrifuge tubes (~~0.1 ml~~ in each), then flash freeze with liquid nitrogen. Store at -80C.	+	#Aliquot into 1.5 ml centrifuge tubes (50uL in each), then flash freeze with liquid nitrogen. Store at -80C.
-	*Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care. See [[Electrocompetent_Cells\| this page]]. ~~Would allow~~ you ~~to calculate efficiency~~.	+	*Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care. See [[Electrocompetent_Cells\| this page]].
		+	* [[User:Janet B. Matsen\|Janet's] cells clump into a "booger" during recovery (pre-plating) that was mostly unspreadable. The clumping is less of an issue if you don't centrifuge them before plating. For this reason I recover in 200 uL and plate all of it.

Janet B. Matsen: /* Electrocompetent Cells */

Janet B. Matsen — Sun, 31 Mar 2013 14:49:11 GMT

Electrocompetent Cells

←Older revision		Revision as of 14:49, 31 March 2013
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	#Use overnight to inoculate: generally use ~ 200 uL/50 mL		#Use overnight to inoculate: generally use ~ 200 uL/50 mL
	#Let grow for about 3-4 h (OD 0.4-0.6 or so), then centrifuge and wash 2-3 times with 10% glycerol (everything on ice).		#Let grow for about 3-4 h (OD 0.4-0.6 or so), then centrifuge and wash 2-3 times with 10% glycerol (everything on ice).
		+	## If using the tube spec, the path length is longer than in the cuvettes. Divide tube specs by 1.65 to convert to the 1 cm path length OD. (If using tube spec, let the OD get to ~1.)
	#Resuspend the pellet in a small volume, ideally up to 1/500<sup>ths</sup> of the culture volume.		#Resuspend the pellet in a small volume, ideally up to 1/500<sup>ths</sup> of the culture volume.
	## See graph below. [[User:Janet B. Matsen\|Janet]] did this experiment. The really concentrated cells performed well despite having clumped into a serious "booger" during recovery (pre-plating) that was mostly unspreadable. [[image:2012_11 electroporation - number of colonies versus competent cell density.jpg\|thumb\|center\|electroporation: number of colonies versus competent cell density]]		## See graph below. [[User:Janet B. Matsen\|Janet]] did this experiment. The really concentrated cells performed well despite having clumped into a serious "booger" during recovery (pre-plating) that was mostly unspreadable. [[image:2012_11 electroporation - number of colonies versus competent cell density.jpg\|thumb\|center\|electroporation: number of colonies versus competent cell density]]
	#Aliquot into 1.5 ml centrifuge tubes (0.1 ml in each), then flash freeze with liquid nitrogen. Store at -80C.		#Aliquot into 1.5 ml centrifuge tubes (0.1 ml in each), then flash freeze with liquid nitrogen. Store at -80C.
	*Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care. See [[Electrocompetent_Cells\| this page]]. Would allow you to calculate efficiency.		*Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care. See [[Electrocompetent_Cells\| this page]]. Would allow you to calculate efficiency.

Janet B. Matsen: /* Electrocompetent Cells */

Janet B. Matsen — Sun, 31 Mar 2013 14:47:19 GMT

Electrocompetent Cells

←Older revision		Revision as of 14:47, 31 March 2013
Line 162:		Line 162:
	#Let grow for about 3-4 h (OD 0.4-0.6 or so), then centrifuge and wash 2-3 times with 10% glycerol (everything on ice).		#Let grow for about 3-4 h (OD 0.4-0.6 or so), then centrifuge and wash 2-3 times with 10% glycerol (everything on ice).
	#Resuspend the pellet in a small volume, ideally up to 1/500<sup>ths</sup> of the culture volume.		#Resuspend the pellet in a small volume, ideally up to 1/500<sup>ths</sup> of the culture volume.
-	## See graph below. [[User:Janet B. Matsen\|Janet]] did this experiment. The really concentrated cells performed well despite having clumped into a serious "booger" during recovery (pre-plating) that was mostly unspreadable.	+	## See graph below. [[User:Janet B. Matsen\|Janet]] did this experiment. The really concentrated cells performed well despite having clumped into a serious "booger" during recovery (pre-plating) that was mostly unspreadable. [[image:2012_11 electroporation - number of colonies versus competent cell density.jpg\|thumb\|center\|electroporation: number of colonies versus competent cell density]]
	#Aliquot into 1.5 ml centrifuge tubes (0.1 ml in each), then flash freeze with liquid nitrogen. Store at -80C.		#Aliquot into 1.5 ml centrifuge tubes (0.1 ml in each), then flash freeze with liquid nitrogen. Store at -80C.
	*Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care. See [[Electrocompetent_Cells\| this page]]. Would allow you to calculate efficiency.		*Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care. See [[Electrocompetent_Cells\| this page]]. Would allow you to calculate efficiency.
-	~~[[image:2012_11 electroporation - number of colonies versus competent cell density.jpg\|thumb\|center\|electroporation: number of colonies versus competent cell density]]~~

Janet B. Matsen: /* CCMB (1L) */

Janet B. Matsen — Fri, 01 Mar 2013 22:42:04 GMT

CCMB (1L)

←Older revision		Revision as of 22:42, 1 March 2013
Line 57:		Line 57:
	* Dispense into smaller bottles for lower contamination risk		* Dispense into smaller bottles for lower contamination risk

-	== CCMB (1L) ==	+	== CCMB (1 Liter) ==

	{\|border="2" cellpadding="1"		{\|border="2" cellpadding="1"

Janet B. Matsen: /* SOB-Mg growth medium: 1L */

Janet B. Matsen — Fri, 01 Mar 2013 22:41:53 GMT

SOB-Mg growth medium: 1L

←Older revision		Revision as of 22:41, 1 March 2013
Line 35:		Line 35:

	====Recipes:====		====Recipes:====
-	== SOB-Mg growth medium~~: 1L~~ ==	+	== SOB-Mg growth medium (1 Liter) ==

	{\|border="2" cellpadding="1"		{\|border="2" cellpadding="1"

Janet B. Matsen: /* SOB-Mg growth medium: 1L */

Janet B. Matsen — Fri, 01 Mar 2013 19:30:54 GMT

SOB-Mg growth medium: 1L

←Older revision		Revision as of 19:30, 1 March 2013
Line 40:		Line 40:
	\|+Setting cell widths		\|+Setting cell widths
	!width="120"\|Ingredient		!width="120"\|Ingredient
-	!width="40"\|Amount	+	!width="60"\|Amount
	\|-		\|-
	\|Bacto Tryptone\|\|20g		\|Bacto Tryptone\|\|20g

Janet B. Matsen: /* Recipes: */

Janet B. Matsen — Fri, 01 Mar 2013 19:30:39 GMT

Recipes:

←Older revision		Revision as of 19:30, 1 March 2013
Line 35:		Line 35:

	====Recipes:====		====Recipes:====
-	*SOB-Mg growth medium	+	== SOB-Mg growth medium: 1L ==
-	*CCMB	+
		+	{\|border="2" cellpadding="1"
		+	\|+Setting cell widths
		+	!width="120"\|Ingredient
		+	!width="40"\|Amount
		+	\|-
		+	\|Bacto Tryptone\|\|20g
		+	\|
		+	\|-
		+	\|Bacto Yeast Extract\|\|5g
		+	\|
		+	\|-
		+	\|1M NaCl\|\|10mL
		+	\|
		+	\|-
		+	\|1M KCl\|\|2.5mL
		+	\|}
		+	* Add water to make 1L
		+	* Autoclave
		+	* Dispense into smaller bottles for lower contamination risk
		+
		+	== CCMB (1L) ==
		+
		+	{\|border="2" cellpadding="1"
		+	\|+Setting cell widths
		+	!width="180"\|Ingredient
		+	!width="60"\|Amount
		+	!width="140"\|Final Concentration
		+	\|-
		+	\|Potassium Acetate, 1M, pH 7\|\|10 mL\|\|10mM
		+	\|
		+	\|-
		+	\|Glycerol\|\|100g\|\|10% (w/v)
		+	\|
		+	\|-
		+	\|CaCl2.2H2O\|\|11.8g\|\|80mM
		+	\|
		+	\|-
		+	\|MnCl2.4H2O\|\|4g\|\|20mM
		+	\|
		+	\|-
		+	\|MgCl2.6H2O\|\|2.5mL\|\|10mM
		+	\|}
		+
		+	# Prepare a 1M solution of potassium acetate, pH 7.0 using KOH.
		+	## Filter through a 0.2 uM membrane & store frozen
		+	# Prepare a solution of 10% potassium acetate, 10% glycerol
		+	# Add salts, allowing each to enter solution before adding the next.
		+	# Adjust pH to 6.4 with 0.1M HCl. Do not adjust pH upward with base.
		+	# Filter through a 0.2 uM filter & store at 4<sup>o</sup>C.

	===Nicole/Andrew protocol for Chemically Competent cells===		===Nicole/Andrew protocol for Chemically Competent cells===

Janet B. Matsen: /* Method */

Janet B. Matsen — Sat, 05 Jan 2013 01:12:26 GMT

Method

←Older revision		Revision as of 01:12, 5 January 2013
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	## Grow cells overnight or several hours in media with appropriate antibiotics if available.		## Grow cells overnight or several hours in media with appropriate antibiotics if available.
	## Use more inoculum in the next step if cultures weren't grown overnight.		## Use more inoculum in the next step if cultures weren't grown overnight.
-	# Inoculate 50 mL SOB-Mg growth medium with this culture. Use ~~between 1~~ uL ~~to 1 mL~~ stationary phase culture ~~per~~ mL of medium.	+	# Inoculate 50 mL SOB-Mg growth medium with this culture. Use 500 uL of stationary phase culture for 50 mL SOB-Mg medium.
	# Incubate at 275 rpm, 37<sup>o</sup>C until OD<sub>600</sub> is about 0.3, which corresponds to ~ 5*10<sup>7</sup>cells/mL		# Incubate at 275 rpm, 37<sup>o</sup>C until OD<sub>600</sub> is about 0.3, which corresponds to ~ 5*10<sup>7</sup>cells/mL
		+	## Higher OD isn't usually a problem for routine work.
	# Collect in sterile 50 mL polypropylene centrifuge tube(s) and chill on ice for 10 minutes		# Collect in sterile 50 mL polypropylene centrifuge tube(s) and chill on ice for 10 minutes
	# Pellet the cells at 750 - 1,000g (2500 rpm) for 14 min at 4<sup>o</sup>C. Decant the supernatant and invert tubes to remove excess culture medium.		# Pellet the cells at 750 - 1,000g (2500 rpm) for 14 min at 4<sup>o</sup>C. Decant the supernatant and invert tubes to remove excess culture medium.
Line 32:		Line 33:
	# Flash freeze with liquid nitrogen		# Flash freeze with liquid nitrogen
	# Store at -80oC to preserve them for many months		# Store at -80oC to preserve them for many months
		+
	====Recipes:====		====Recipes:====
	*SOB-Mg growth medium		*SOB-Mg growth medium

Janet B. Matsen at 02:21, 18 November 2012

Janet B. Matsen — Sun, 18 Nov 2012 02:21:26 GMT

←Older revision		Revision as of 02:21, 18 November 2012
Line 9:		Line 9:
	*You need to flash freeze the cells at the end of the procedure. You can do this by pouring liquid nitrogen over them, or you can freeze your tubes at -80oC the night before, put your aliquots in, and stick them back at -80oC.		*You need to flash freeze the cells at the end of the procedure. You can do this by pouring liquid nitrogen over them, or you can freeze your tubes at -80oC the night before, put your aliquots in, and stick them back at -80oC.

-	==Amanda/Janet Protocol for Chemically Competent Cells ==	+	===Amanda/Janet Protocol for Chemically Competent Cells ===
-	=== Supplies needed: ===	+	==== Supplies needed: ====
	* Plate of E. Coli colonies		* Plate of E. Coli colonies
	* SOB-Mg growth medium		* SOB-Mg growth medium
Line 17:		Line 17:
	* Freezer tubes (1.5 mL)		* Freezer tubes (1.5 mL)
	* SOC medium for recovery after heat shock (LB/TB is fine instead)		* SOC medium for recovery after heat shock (LB/TB is fine instead)
-	=== Method ===	+	==== Method ====
	# Pick several colonies off a freshly streaked plate into ~1 mL SOB-Mg growth medium		# Pick several colonies off a freshly streaked plate into ~1 mL SOB-Mg growth medium
	## Grow cells overnight or several hours in media with appropriate antibiotics if available.		## Grow cells overnight or several hours in media with appropriate antibiotics if available.
Line 32:		Line 32:
	# Flash freeze with liquid nitrogen		# Flash freeze with liquid nitrogen
	# Store at -80oC to preserve them for many months		# Store at -80oC to preserve them for many months
-	===Recipes:===	+	====Recipes:====
	*SOB-Mg growth medium		*SOB-Mg growth medium
	*CCMB		*CCMB

-	==Nicole/Andrew protocol for Chemically Competent cells==	+	===Nicole/Andrew protocol for Chemically Competent cells===
	'''Materials and reagents'''		'''Materials and reagents'''
	* E. coli line (Top 10, S17-1, BL21-AL, BL21-D3, JM109, Qiagen)		* E. coli line (Top 10, S17-1, BL21-AL, BL21-D3, JM109, Qiagen)
Line 107:		Line 107:
	==Electrocompetent Cells==		==Electrocompetent Cells==
	*You can make your own electro-competent cells for electroporation.		*You can make your own electro-competent cells for electroporation.
-	**From Amada's ~~past mentor in undergrad:~~	+	#Grow a small (~ 2 mL) overnight culture with appropriate antibiotic(s)
-	***Grow the cells overnight	+	#Use overnight to inoculate: generally use ~ 200 uL/50 mL
-	***Inoculate from this the next morning: generally use 200 uL/50 mL	+	#Let grow for about 3-4 h (OD 0.4-0.6 or so), then centrifuge and wash 2-3 times with 10% glycerol (everything on ice).
-	~~***~~Let grow for about 3-4 h (OD 0.4-0.6 or so), then centrifuge and wash 3 times with 10% glycerol (everything on ice).	+	#Resuspend the pellet in a small volume, ideally up to 1/500<sup>ths</sup> of the culture volume.
-	~~***~~Resuspend the pellet in a small volume ~~(Example~~, ~~if I do a 5 ml culture I try~~ to ~~resuspend~~ the ~~final pellet in no more than 0~~.~~2 or 0~~.~~3 ml of 10% glycerol~~). ~~Then split in~~ 1.5 ml centrifuge tubes (0.1 ml in each), freeze ~~on dry ice-isopropanol and store~~ at -80C.	+	## See graph below. [[User:Janet B. Matsen\|Janet]] did this experiment. The really concentrated cells performed well despite having clumped into a serious "booger" during recovery (pre-plating) that was mostly unspreadable.
-	~~**~~Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care. See [[Electrocompetent_Cells\| this page]]. Would allow you to calculate efficiency.	+	#Aliquot into 1.5 ml centrifuge tubes (0.1 ml in each), then flash freeze with liquid nitrogen. Store at -80C.
		+	*Note: you can dilute your re-suspension, measure OD, and calculate the cell concentration if you care. See [[Electrocompetent_Cells\| this page]]. Would allow you to calculate efficiency.
		+	[[image:2012_11 electroporation - number of colonies versus competent cell density.jpg\|thumb\|center\|electroporation: number of colonies versus competent cell density]]

Janet B. Matsen: /* Amanda/Janet Protocol for Chemically Competent Cells */

Janet B. Matsen — Sat, 20 Oct 2012 01:08:59 GMT

Amanda/Janet Protocol for Chemically Competent Cells

←Older revision		Revision as of 01:08, 20 October 2012
Line 16:		Line 16:
	* 50 mL screw-cap polypropylene tubes		* 50 mL screw-cap polypropylene tubes
	* Freezer tubes (1.5 mL)		* Freezer tubes (1.5 mL)
-	* SOC medium (LB/TB is fine instead)	+	* SOC medium for recovery after heat shock (LB/TB is fine instead)
	=== Method ===		=== Method ===
-	# Pick several colonies off a freshly streaked plate into 1 mL SOB-Mg growth medium	+	# Pick several colonies off a freshly streaked plate into ~1 mL SOB-Mg growth medium
-	## Grow overnight in media with appropriate antibiotics if available	+	## Grow cells overnight or several hours in media with appropriate antibiotics if available.
		+	## Use more inoculum in the next step if cultures weren't grown overnight.
	# Inoculate 50 mL SOB-Mg growth medium with this culture. Use between 1 uL to 1 mL stationary phase culture per mL of medium.		# Inoculate 50 mL SOB-Mg growth medium with this culture. Use between 1 uL to 1 mL stationary phase culture per mL of medium.
-	#	+	# Incubate at 275 rpm, 37<sup>o</sup>C until OD<sub>600</sub> is about 0.3, which corresponds to ~ 5*10<sup>7</sup>cells/mL
-		+	# Collect in sterile 50 mL polypropylene centrifuge tube(s) and chill on ice for 10 minutes
-		+	# Pellet the cells at 750 - 1,000g (2500 rpm) for 14 min at 4<sup>o</sup>C. Decant the supernatant and invert tubes to remove excess culture medium.
		+	# Disperse cells in ~1/3 volume of CCMB by gentle vortexing or rapping of the centrifuge tube.
		+	# Incubate on ice for 20 minutes
		+	# Centrifuge at 2500 rpm for 10 min at 4<sup>o</sup>C
		+	# Resuspend cells in CCMB at 1/12 the original culture volume
		+	# Make aliquots in eppendorf tubes, ideally on ice
		+	# Flash freeze with liquid nitrogen
		+	# Store at -80oC to preserve them for many months
		+	===Recipes:===
		+	*SOB-Mg growth medium
		+	*CCMB

	==Nicole/Andrew protocol for Chemically Competent cells==		==Nicole/Andrew protocol for Chemically Competent cells==