Richard Lab:Electroporation of E. coli - Revision history

Michael A. Speer: /* Procedure */

Michael A. Speer — Wed, 06 Apr 2011 21:11:11 GMT

Procedure

←Older revision		Revision as of 21:11, 6 April 2011
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	2. Remove # vials containing 100μl electro-competent cells from the -80°C freezer and thaw them with the iced cuvettes.<br>		2. Remove # vials containing 100μl electro-competent cells from the -80°C freezer and thaw them with the iced cuvettes.<br>
	3. Prepare # micro-centrifuge tubes containing 900μl SOB media.<br>		3. Prepare # micro-centrifuge tubes containing 900μl SOB media.<br>
-	4. Turn on electroporator and set voltage to ~~either~~ 1.25 kV (1mm cuvettes).<br>	+	4. Turn on electroporator and set voltage to 1.5 kV (1mm cuvettes).<br>
	5. Add add 5μL of ligated DNA sample to 100μl thawed electrocompetent cells on ice. Swirl tip around gently in cells to mix DNA and cells.<br>		5. Add add 5μL of ligated DNA sample to 100μl thawed electrocompetent cells on ice. Swirl tip around gently in cells to mix DNA and cells.<br>
	6. Place cells back on ice to ensure they remain cold.<br>		6. Place cells back on ice to ensure they remain cold.<br>
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	11. Quickly use a pipette to remove the electroporated cell suspension from the cuvette and add it to a tubes containing 1ml SOB.<br>		11. Quickly use a pipette to remove the electroporated cell suspension from the cuvette and add it to a tubes containing 1ml SOB.<br>
	12. Let cells recover at room temperature for 30-60 minutes.<br>		12. Let cells recover at room temperature for 30-60 minutes.<br>
-	13. Plate 100μl of eletroporated cells onto prewarmed LB-agar plate supplemented with appropriate antibiotic. Incubate plate overnight at 37°C.<br>	+	13. Plate 100μl of eletroporated cells onto prewarmed LB-agar plate supplemented with appropriate antibiotic. Incubate plate overnight at 37°C.<br>
-		+

	===Notes===		===Notes===

Michael A. Speer at 00:41, 13 January 2011

Michael A. Speer — Thu, 13 Jan 2011 00:41:03 GMT

←Older revision		Revision as of 00:41, 13 January 2011
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	Back to [[Richard_Lab:protocols \| Protocols]]		Back to [[Richard_Lab:protocols \| Protocols]]
-
-	~~[[Image:Gel_electrophoresis.JPG\|thumb\|center\|300px]]~~

	<center>		<center>

Michael A. Speer at 18:48, 23 June 2009

Michael A. Speer — Tue, 23 Jun 2009 18:48:42 GMT

←Older revision		Revision as of 18:48, 23 June 2009
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	*Other protocols (specificially the [[Knight:Electroporation\|Knight Protocol]]) were used extensively in the development of this protocol and have much more accessory information than this protocol contains. Feel free to access these protocols from the [[Electroporation\|consensus protocol page]]		*Other protocols (specificially the [[Knight:Electroporation\|Knight Protocol]]) were used extensively in the development of this protocol and have much more accessory information than this protocol contains. Feel free to access these protocols from the [[Electroporation\|consensus protocol page]]

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

-	Back to [[Richard_Lab \| ~~Richard Lab~~]]	+
		+	Back to [[Richard_Lab:protocols \| Protocols]]

Michael A. Speer: /* Notes */

Michael A. Speer — Tue, 23 Jun 2009 18:46:40 GMT

Notes

←Older revision		Revision as of 18:46, 23 June 2009
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	===Notes===		===Notes===
	* When choosing the # of vials and cuvettes, be sure to include 2 vials for two negative controls (one with no DNA added, and another with only cut vector added).		* When choosing the # of vials and cuvettes, be sure to include 2 vials for two negative controls (one with no DNA added, and another with only cut vector added).
-	*Other protocols (~~Specificially~~ the [[Knight:Electroporation\|Knight Protocol]] were used extensively in the development of this protocol.	+	*Other protocols (specificially the [[Knight:Electroporation\|Knight Protocol]]) were used extensively in the development of this protocol and have much more accessory information than this protocol contains. Feel free to access these protocols from the [[Electroporation\|consensus protocol page]]

	[[Category:Protocol]] [[Category:In vivo]] [[Category:DNA]]		[[Category:Protocol]] [[Category:In vivo]] [[Category:DNA]]

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Michael A. Speer: /* Notes */

Michael A. Speer — Tue, 23 Jun 2009 18:44:41 GMT

Notes

←Older revision		Revision as of 18:44, 23 June 2009
Line 25:		Line 25:
	===Notes===		===Notes===
	* When choosing the # of vials and cuvettes, be sure to include 2 vials for two negative controls (one with no DNA added, and another with only cut vector added).		* When choosing the # of vials and cuvettes, be sure to include 2 vials for two negative controls (one with no DNA added, and another with only cut vector added).
-	Other protocols (Specificially the [[Knight:Electroporation\|Knight Protocol]] were used extensively in the development of this protocol.	+	*Other protocols (Specificially the [[Knight:Electroporation\|Knight Protocol]] were used extensively in the development of this protocol.

	[[Category:Protocol]] [[Category:In vivo]] [[Category:DNA]]		[[Category:Protocol]] [[Category:In vivo]] [[Category:DNA]]

	Back to [[Richard_Lab \| Richard Lab]]		Back to [[Richard_Lab \| Richard Lab]]

Michael A. Speer at 18:44, 23 June 2009

Michael A. Speer — Tue, 23 Jun 2009 18:44:17 GMT

Michael A. Speer at 18:40, 23 June 2009

Michael A. Speer — Tue, 23 Jun 2009 18:40:00 GMT

←Older revision		Revision as of 18:40, 23 June 2009
Line 8:		Line 8:

	===Procedure===		===Procedure===
-		+	1. Chill the # electroporation cuvettes by floating them in an ice bath.
-	# Chill electroporation cuvettes ~~and DNA samples on~~ ice.	+	2. Remove # vials containing 100μl electro-competent cells from the -80°C freezer and thaw them with the iced cuvettes.
-	# Remove # vials containing 100μl electro-competent cells from the -80°C freezer~~. Thaw~~ the ~~vials on ice~~. ~~Be sure to include 2 vials for a negative and an unligated vector control~~.	+	3. Prepare # micro-centrifuge tubes containing 900μl SOB media.
-	# Prepare # micro-centrifuge tubes containing 900μl SOB media.	+	4. Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes)
-	# Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes)	+	5. Add add 5μL of ligated DNA sample to 100μl thawed electrocompetent cells on ice. Swirl tip around gently in cells to mix DNA and cells.
-	# Add add 5μL of ligated DNA sample to 100μl thawed electrocompetent cells on ice. Swirl tip around gently in cells to mix DNA and cells.	+	6. Place cells back on ice to ensure they remain cold.
-	# Place cells back on ice to ensure they remain cold.	+	7. Pippette 100μL of cell-DNA mixture to cuvette.
-	# Pippette 100μL of cell-DNA mixture to cuvette.	+	8. Wipe off excess moisture from outside of cuvette.
-	# Wipe off excess moisture from outside of cuvette.	+	9. Place cuvette in chamber of electroporator.
-	# Place cuvette in chamber of electroporator.	+	10. Pulse the cells by pressing button on electroporator twice.
-	# Pulse the cells by pressing button on electroporator twice.	+	11. Quickly use a pipette to remove the electroporated cell suspension from the cuvette and add it to a tubes containing 1ml SOB.
-	# Quickly use a pipette to remove the electroporated cell suspension from the cuvette and add it to a tubes containing 1ml SOB.	+	12. Let cells recover at room temperature for 30-60 minutes.
-	# Let cells recover at room temperature for 30-60 minutes.	+	13. Plate 100μl of eletroporated cells onto prewarmed LB-agar plate supplemented with appropriate antibiotic. Incubate plate overnight at 37°C.
-	# Plate 100μl of eletroporated cells onto prewarmed LB-agar plate supplemented with appropriate antibiotic. Incubate plate overnight at 37°C.	+	14. Leave remaining SOB-cell mixture on the bench-top overnight.
-	# Leave remaining SOB-cell mixture on the bench-top overnight.	+	15. If you don't have any transformants in the morning then plate the rest of the transformation.
-	# If you don't have any transformants in the morning then plate the rest of the transformation.	+

	===Notes===		===Notes===
-	* ~~Our "1X TBE" is technically 0.5X TBE (5.3g/L Tris Base~~, 2~~.75g/L Boric Acid~~, and ~~2.9g/L EDTA~~) ~~but for our purposes we call it 1X TBE~~.	+	* When choosing the # of vials and cuvettes, be sure to include 2 vials for two negative controls (one with no DNA added, and another with only cut vector added).
-	** Correspondingly, the ~~10X TBE is officially 5X.~~	+	Other protocols (Specificially the [[Knight Protocol]] were used extensively in the development of this protocol.
-	*Using TBE allows us to have such a high (150V) voltage. If you use TAE you need to use a significantly lower voltage and your run time will be longer. Despite that, TAE is advantagous in some cases, but [[~~User:Michael_A. Speer \| Mike~~]] ~~feels that TBE is better suited for his applications. This is discussed in detail~~ in the ~~[[agarose gel electrophoresis \| consensus~~ protocol]].	+
-	* Use the following table to determine the amount of agarose you want to use.	+	[[Category:Protocol]] [[Category:In vivo]] [[Category:DNA]]
-	~~<center>~~	+
-	~~{\| border="1" cellpadding="5" cellspacing="0" align="center"~~	+
-	~~\|+ '''Choosing an Agarose Concentration'''~~	+
-	~~! Agarose Concentration (g/100mL) !! Optimal DNA Resolution (kb)~~	+
-	~~\|-=~~	+
-	~~\| align="center"\|0.5 \|\| align="center"\|1 - 30~~	+
-	\|-	+
-	~~\| align="center"\|0.7 \|\| align="center"\|0.8 - 12~~	+
-	\|-	+
-	~~\| align="center"\|1.0 \|\| align="center"\|0.5 - 10~~	+
-	\|-	+
-	~~\| align="center"\|1.2 \|\| align="center"\|0.4 - 7~~	+
-	\|-	+
-	~~\| align="center"\|1.5 \|\| align="center"\|0.2 - 3~~	+
-	\|}	+
-	~~</center>~~	+
-	[[Category:Protocol]] [[Category:In ~~vitro~~]] [[Category:DNA]]	+

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Michael A. Speer: New page: Back to Protocols 300px '''This protocol is for the typical electro-transformation of ''E. coli'' done...

Michael A. Speer — Tue, 23 Jun 2009 18:32:33 GMT

New page: Back to Protocols 300px <center> '''This protocol is for the typical electro-transformation of ''E. coli'' done...

New page

Back to [[Richard_Lab:protocols | Protocols]]

[[Image:Gel_electrophoresis.JPG|thumb|center|300px]]

<center>
'''This protocol is for the typical electro-transformation of ''E. coli'' done in the Richard Lab.
'''The [[Electroporation | consensus electroporation protocol]] should be consulted if deviating from the procedure outlined here.'''</center>

===Procedure===

# Chill electroporation cuvettes and DNA samples on ice.
# Remove # vials containing 100μl electro-competent cells from the -80°C freezer. Thaw the vials on ice. Be sure to include 2 vials for a negative and an unligated vector control.
# Prepare # micro-centrifuge tubes containing 900μl SOB media.
# Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes)
# Add add 5μL of ligated DNA sample to 100μl thawed electrocompetent cells on ice. Swirl tip around gently in cells to mix DNA and cells.
# Place cells back on ice to ensure they remain cold.
# Pippette 100μL of cell-DNA mixture to cuvette.
# Wipe off excess moisture from outside of cuvette.
# Place cuvette in chamber of electroporator.
# Pulse the cells by pressing button on electroporator twice.
# Quickly use a pipette to remove the electroporated cell suspension from the cuvette and add it to a tubes containing 1ml SOB.
# Let cells recover at room temperature for 30-60 minutes.
# Plate 100μl of eletroporated cells onto prewarmed LB-agar plate supplemented with appropriate antibiotic. Incubate plate overnight at 37°C.
# Leave remaining SOB-cell mixture on the bench-top overnight.
# If you don't have any transformants in the morning then plate the rest of the transformation.

===Notes===
* Our "1X TBE" is technically 0.5X TBE (5.3g/L Tris Base, 2.75g/L Boric Acid, and 2.9g/L EDTA) but for our purposes we call it 1X TBE.
** Correspondingly, the 10X TBE is officially 5X.
*Using TBE allows us to have such a high (150V) voltage. If you use TAE you need to use a significantly lower voltage and your run time will be longer. Despite that, TAE is advantagous in some cases, but [[User:Michael_A. Speer | Mike]] feels that TBE is better suited for his applications. This is discussed in detail in the [[agarose gel electrophoresis | consensus protocol]].
* Use the following table to determine the amount of agarose you want to use.
<center>
{| border="1" cellpadding="5" cellspacing="0" align="center"
|+ '''Choosing an Agarose Concentration'''
! Agarose Concentration (g/100mL) !! Optimal DNA Resolution (kb)
|-=
| align="center"|0.5 || align="center"|1 - 30
|-
| align="center"|0.7 || align="center"|0.8 - 12
|-
| align="center"|1.0 || align="center"|0.5 - 10
|-
| align="center"|1.2 || align="center"|0.4 - 7
|-
| align="center"|1.5 || align="center"|0.2 - 3
|}
</center>
[[Category:Protocol]] [[Category:In vitro]] [[Category:DNA]]

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