Knight:Preparing electrocompetent cells - Revision history

Tk: /* Chemicals and reagents */

Tk — Sat, 05 Apr 2008 20:30:37 GMT

Chemicals and reagents

←Older revision		Revision as of 20:30, 5 April 2008
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	*~500 mL LB Lennox supplemented with appropriate concentration of antibiotic if appropriate.		*~500 mL LB Lennox supplemented with appropriate concentration of antibiotic if appropriate.
	*~600 mL sterile deionized water chilled to 4°C		*~600 mL sterile deionized water chilled to 4°C
-	*~~~22~~ mL sterile 15% glycerol in deionized water chilled to 4°C	+	* 50 mL sterile 10% glycerol in deionized water chilled to 4°C
	*Ice bucket and ice		*Ice bucket and ice
	*Dry ice, ethanol bath		*Dry ice, ethanol bath
		+
	===Supplies===		===Supplies===
	*Many 1.5 mL plastic tubes chilled to -80 °C		*Many 1.5 mL plastic tubes chilled to -80 °C

Tk: /* Supplies */

Tk — Sat, 05 Apr 2008 20:30:11 GMT

Supplies

←Older revision		Revision as of 20:30, 5 April 2008
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	*Dry ice, ethanol bath		*Dry ice, ethanol bath
	===Supplies===		===Supplies===
-	*Many 0.6 mL plastic tubes chilled to -80 °C	+	*Many 1.5 mL plastic tubes chilled to -80 °C
	*14 mL culture tube for starter culture		*14 mL culture tube for starter culture
	*2 L flask for culture		*2 L flask for culture
	*225 mL plastic tubes for centrifugation		*225 mL plastic tubes for centrifugation
	*Pipets		*Pipets
-	*Sucking pipet

	==Procedure==		==Procedure==

Tk: /* Equipment */

Tk — Sat, 05 Apr 2008 20:29:15 GMT

Equipment

←Older revision		Revision as of 20:29, 5 April 2008
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	*-80°C freezer		*-80°C freezer
	*37°C incubator		*37°C incubator
-	*Refrigerated centrifuge that accepts 225 mL culture tubes	+	*Refrigerated centrifuge that accepts 225 mL culture tubes
-	*Refrigerated centrifuge that accepts 1.5 mL tubes	+
	===Chemicals and reagents===		===Chemicals and reagents===
	*~500 mL LB Lennox supplemented with appropriate concentration of antibiotic if appropriate.		*~500 mL LB Lennox supplemented with appropriate concentration of antibiotic if appropriate.

Tk: /* Procedure */

Tk — Sat, 05 Apr 2008 20:29:00 GMT

Procedure

←Older revision		Revision as of 20:29, 5 April 2008
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	# Remove supernatant and gently resuspend pellets with 200mL cold sterile water. <br> Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water.		# Remove supernatant and gently resuspend pellets with 200mL cold sterile water. <br> Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water.
	# Centrifuge for 15 mins at 2000g at 4°C		# Centrifuge for 15 mins at 2000g at 4°C
-	# Remove supernatant and gently resuspend pellets with ~~100mL~~ cold sterile water. <br> Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water.	+	# Remove supernatant and gently resuspend pellets with 200mL cold sterile water. <br> Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water.
		+	# Hold on ice for 30 minutes
	# Centrifuge for 15 mins at 2000g at 4°C		# Centrifuge for 15 mins at 2000g at 4°C
-	# Remove supernatant and gently resuspend pellets with ~~10mL~~ cold 15% glycerol. <br> This can be optionally transferred to a 14 mL conical tube.	+	# Remove supernatant and gently resuspend pellets with 25mL cold 10% glycerol. <br> This can be optionally transferred to a 50 mL conical tube.
		+	# Hold on ice for 30 minutes
	# Centrifuge for 15 mins at 1500g at 4°C		# Centrifuge for 15 mins at 1500g at 4°C
-	# Remove supernatant and ~~transfer pellet to 1.5 mL tube using sucking pipet. <br> There should be sufficient glycerol in the pellet for you to do this.~~	+	# Remove the supernatant and add 500 μl of 10% glycerol
-	~~# Microcentrifuge 5 mins at maximum speed at 4~~&~~deg~~;C.	+	# Resuspend the cells in a final volume of approximately 1 ml
-	~~# Remove supernatant.~~	+	# Aliquot 50 μL per tube (tubes on ice)
-	# Resuspend ~~pellet~~ in ~~1mL cold 15% glycerol.~~	+
-	# Aliquot 50 μL per tube.	+
	# Shock freeze cell suspensions in a dry ice and ethanol bath. <br> One website recommended against using liquid nitrogen but did not justify this recommendation.		# Shock freeze cell suspensions in a dry ice and ethanol bath. <br> One website recommended against using liquid nitrogen but did not justify this recommendation.
	# Store at -80°C		# Store at -80°C

Tk: /* Procedure */

Tk — Sat, 05 Apr 2008 20:22:08 GMT

Procedure

←Older revision		Revision as of 20:22, 5 April 2008
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	#Inoculate 5mL LB medium and grow overnight at 37°C with rotation. <br> Use LB Lennox rather than LB Miller in order to lessen salt content of media		#Inoculate 5mL LB medium and grow overnight at 37°C with rotation. <br> Use LB Lennox rather than LB Miller in order to lessen salt content of media
	# Add the 5mL overnight culture to 450mL LB medium and incubate at 37°C with vigorous shaking until the OD 600nm is between 0.5 and 1.0. It should take about 3 hours. <br> For recA<sup>-</sup> strains, the OD 600 nm should be between 0.5 and 0.7 according to one online source.		# Add the 5mL overnight culture to 450mL LB medium and incubate at 37°C with vigorous shaking until the OD 600nm is between 0.5 and 1.0. It should take about 3 hours. <br> For recA<sup>-</sup> strains, the OD 600 nm should be between 0.5 and 0.7 according to one online source.
		+	# Fast cool the centrifuge with the correct rotor to 4°C
	# Pour the culture into two 225 mL centrifuge tubes.		# Pour the culture into two 225 mL centrifuge tubes.
	# Place the tubes on ice for 15 minutes. <br> This step can vary in incubation time between 15 minutes and 1 hr. Longer incubation times may lead to higher competency.<br>''For the following steps it is important to keep cells cold and remove all the supernatant in each step to remove residual ions.''<br>		# Place the tubes on ice for 15 minutes. <br> This step can vary in incubation time between 15 minutes and 1 hr. Longer incubation times may lead to higher competency.<br>''For the following steps it is important to keep cells cold and remove all the supernatant in each step to remove residual ions.''<br>

Tk: /* Procedure */

Tk — Sat, 05 Apr 2008 20:20:16 GMT

Procedure

←Older revision		Revision as of 20:20, 5 April 2008
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	==Procedure==		==Procedure==

-	#Prechill all tubes and pipets at 4°C or -80°C as appropriate. <br> Also rinse all flasks with H<sub>2</sub>O prior to autoclaving in order to remove residual detergents that may remain on glassware from dishwashing. This step may increase competency.	+	#Prechill all tubes and pipets at 4°C or -80°C as appropriate. <br> Also rinse all flasks with H<sub>2</sub>O prior to autoclaving in order to remove residual detergents that may remain on glassware from dishwashing. This step may increase competency. Autoclaving with water, which is then discarded, is even better.
	#Inoculate 5mL LB medium and grow overnight at 37°C with rotation. <br> Use LB Lennox rather than LB Miller in order to lessen salt content of media		#Inoculate 5mL LB medium and grow overnight at 37°C with rotation. <br> Use LB Lennox rather than LB Miller in order to lessen salt content of media
	# Add the 5mL overnight culture to 450mL LB medium and incubate at 37°C with vigorous shaking until the OD 600nm is between 0.5 and 1.0. It should take about 3 hours. <br> For recA<sup>-</sup> strains, the OD 600 nm should be between 0.5 and 0.7 according to one online source.		# Add the 5mL overnight culture to 450mL LB medium and incubate at 37°C with vigorous shaking until the OD 600nm is between 0.5 and 1.0. It should take about 3 hours. <br> For recA<sup>-</sup> strains, the OD 600 nm should be between 0.5 and 0.7 according to one online source.

Reshma P. Shetty at 21:20, 8 May 2007

Reshma P. Shetty — Tue, 08 May 2007 21:20:33 GMT

←Older revision		Revision as of 21:20, 8 May 2007
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	*'''[[User:Rshetty\|RS]] 17:49, 13 June 2006 (EDT)''': Dont' forget to use the conical bottoms on the swinging buckets in the centrifuge ... not the flat ones. Otherwise, your tubes will break. (Oops.)		*'''[[User:Rshetty\|RS]] 17:49, 13 June 2006 (EDT)''': Dont' forget to use the conical bottoms on the swinging buckets in the centrifuge ... not the flat ones. Otherwise, your tubes will break. (Oops.)

-	[[Category:~~Escherichia coli~~]] [[Category:~~Protocol~~]]	+	[[Category:Protocol]]
		+	[[Category:In vivo]]
		+	[[Category:Escherichia coli]]

Reshma P. Shetty at 17:28, 20 April 2007

Reshma P. Shetty — Fri, 20 Apr 2007 17:28:46 GMT

←Older revision		Revision as of 17:28, 20 April 2007
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	==Notes==		==Notes==
	*'''[[User:Rshetty\|RS]] 17:49, 13 June 2006 (EDT)''': Dont' forget to use the conical bottoms on the swinging buckets in the centrifuge ... not the flat ones. Otherwise, your tubes will break. (Oops.)		*'''[[User:Rshetty\|RS]] 17:49, 13 June 2006 (EDT)''': Dont' forget to use the conical bottoms on the swinging buckets in the centrifuge ... not the flat ones. Otherwise, your tubes will break. (Oops.)
		+
		+	[[Category:Escherichia coli]] [[Category:Protocol]]

ClarkeS: spelling

ClarkeS — Thu, 20 Jul 2006 22:23:41 GMT

spelling

←Older revision		Revision as of 22:23, 20 July 2006
Line 8:		Line 8:
	*-80°C freezer		*-80°C freezer
	*37°C incubator		*37°C incubator
-	*~~Refridgerated~~ centrifuge that accepts 225 mL culture tubes	+	*Refrigerated centrifuge that accepts 225 mL culture tubes
-	*~~Refridgerated~~ centrifuge that accepts 1.5 mL tubes	+	*Refrigerated centrifuge that accepts 1.5 mL tubes
	===Chemicals and reagents===		===Chemicals and reagents===
	*~500 mL LB Lennox supplemented with appropriate concentration of antibiotic if appropriate.		*~500 mL LB Lennox supplemented with appropriate concentration of antibiotic if appropriate.

Reshma P. Shetty at 21:49, 13 June 2006

Reshma P. Shetty — Tue, 13 Jun 2006 21:49:28 GMT

←Older revision		Revision as of 21:49, 13 June 2006
Line 45:		Line 45:
	# Shock freeze cell suspensions in a dry ice and ethanol bath. <br> One website recommended against using liquid nitrogen but did not justify this recommendation.		# Shock freeze cell suspensions in a dry ice and ethanol bath. <br> One website recommended against using liquid nitrogen but did not justify this recommendation.
	# Store at -80°C		# Store at -80°C
		+
		+	==Notes==
		+	*'''[[User:Rshetty\|RS]] 17:49, 13 June 2006 (EDT)''': Dont' forget to use the conical bottoms on the swinging buckets in the centrifuge ... not the flat ones. Otherwise, your tubes will break. (Oops.)