Talk:TOP10 chemically competent cells - Revision history

Tk at 22:26, 17 February 2013

2013-02-17T22:26:11Z

←Older revision		Revision as of 22:26, 17 February 2013
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	==Questions==		==Questions==
	*'''[[User:Bcanton\|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?		*'''[[User:Bcanton\|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?
		+
		+	Please do not modify this protocol without discussion on this talk page.

Andy Lane: /* Transformation efficiencies */

2010-11-26T21:49:36Z

Transformation efficiencies

←Older revision		Revision as of 21:49, 26 November 2010
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	==Transformation efficiencies==		==Transformation efficiencies==
	*'''[[User:Reshma P. Shetty\|Reshma]] 11:36, 12 June 2007 (EDT)''': Using this protocol (including the measurement of competence), I obtained efficiencies of 3E8 for TOP10 cells.		*'''[[User:Reshma P. Shetty\|Reshma]] 11:36, 12 June 2007 (EDT)''': Using this protocol (including the measurement of competence), I obtained efficiencies of 3E8 for TOP10 cells.
-	*'''[[User:Andy Lane\|Andy Lane]] 16:33, 26 November 2010 (EST):''' I achieved competencies of 8E7 for DH5a cells and 5.5E7 for XL-10 Gold cells. DH5a ~~was~~ harvested at OD600 of 0.3; XL-10 Gold at 0.35) However, I lost many cells during the centrifugation steps and froze down at a lower density than recommended. The protocol states that a 1:5 dilution in SOC should have an OD600 of 1-1.5 at freezing; mine was only 0.55 - 0.6 for both strains.	+	*'''[[User:Andy Lane\|Andy Lane]] 16:33, 26 November 2010 (EST):''' I achieved competencies of 8E7 for DH5a cells and 5.5E7 for XL-10 Gold cells. DH5a were harvested at OD600 of 0.3; XL-10 Gold at 0.35. However, I lost many cells during the centrifugation steps and froze down at a lower density than recommended. The protocol states that a 1:5 dilution in SOC should have an OD600 of 1-1.5 at freezing; mine was only 0.55 - 0.6 for both strains.

	==Questions==		==Questions==
	*'''[[User:Bcanton\|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?		*'''[[User:Bcanton\|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?

Andy Lane: /* Transformation efficiencies */

2010-11-26T21:34:54Z

Transformation efficiencies

←Older revision		Revision as of 21:34, 26 November 2010
Line 18:		Line 18:
	==Transformation efficiencies==		==Transformation efficiencies==
	*'''[[User:Reshma P. Shetty\|Reshma]] 11:36, 12 June 2007 (EDT)''': Using this protocol (including the measurement of competence), I obtained efficiencies of 3E8 for TOP10 cells.		*'''[[User:Reshma P. Shetty\|Reshma]] 11:36, 12 June 2007 (EDT)''': Using this protocol (including the measurement of competence), I obtained efficiencies of 3E8 for TOP10 cells.
-	*'''[[User:Andy Lane\|Andy Lane]]~~'''~~ 16:33, 26 November 2010 (EST) I achieved competencies of 8E7 for DH5a cells and 5.5E7 for XL-10 Gold cells. DH5a was harvested at OD600 of 0.3; XL-10 Gold at 0.35) However, I lost many cells during the centrifugation steps and froze down at a lower density than recommended. The protocol states that a 1:5 dilution in SOC should have an OD600 of 1-1.5 at freezing; mine was only 0.55 - 0.6 for both strains.	+	*'''[[User:Andy Lane\|Andy Lane]] 16:33, 26 November 2010 (EST):''' I achieved competencies of 8E7 for DH5a cells and 5.5E7 for XL-10 Gold cells. DH5a was harvested at OD600 of 0.3; XL-10 Gold at 0.35) However, I lost many cells during the centrifugation steps and froze down at a lower density than recommended. The protocol states that a 1:5 dilution in SOC should have an OD600 of 1-1.5 at freezing; mine was only 0.55 - 0.6 for both strains.

	==Questions==		==Questions==
	*'''[[User:Bcanton\|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?		*'''[[User:Bcanton\|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?

Andy Lane: /* Transformation efficiencies */

2010-11-26T21:34:17Z

Transformation efficiencies

←Older revision		Revision as of 21:34, 26 November 2010
Line 18:		Line 18:
	==Transformation efficiencies==		==Transformation efficiencies==
	*'''[[User:Reshma P. Shetty\|Reshma]] 11:36, 12 June 2007 (EDT)''': Using this protocol (including the measurement of competence), I obtained efficiencies of 3E8 for TOP10 cells.		*'''[[User:Reshma P. Shetty\|Reshma]] 11:36, 12 June 2007 (EDT)''': Using this protocol (including the measurement of competence), I obtained efficiencies of 3E8 for TOP10 cells.
-	--[[User:Andy Lane\|Andy Lane]] 16:33, 26 November 2010 (EST) I achieved competencies of 8E7 for DH5a cells and 5.5E7 for XL-10 Gold cells. DH5a was harvested at OD600 of 0.3; XL-10 Gold at 0.35) However, I lost many cells during the centrifugation steps and froze down at a lower density than recommended. The protocol states that a 1:5 dilution in SOC should have an OD600 of 1-1.5 at freezing; mine was only 0.55 - 0.6 for both strains.	+	*'''[[User:Andy Lane\|Andy Lane]]''' 16:33, 26 November 2010 (EST) I achieved competencies of 8E7 for DH5a cells and 5.5E7 for XL-10 Gold cells. DH5a was harvested at OD600 of 0.3; XL-10 Gold at 0.35) However, I lost many cells during the centrifugation steps and froze down at a lower density than recommended. The protocol states that a 1:5 dilution in SOC should have an OD600 of 1-1.5 at freezing; mine was only 0.55 - 0.6 for both strains.

	==Questions==		==Questions==
	*'''[[User:Bcanton\|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?		*'''[[User:Bcanton\|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?

Andy Lane: /* Transformation efficiencies */

2010-11-26T21:33:45Z

Transformation efficiencies

←Older revision		Revision as of 21:33, 26 November 2010
Line 18:		Line 18:
	==Transformation efficiencies==		==Transformation efficiencies==
	*'''[[User:Reshma P. Shetty\|Reshma]] 11:36, 12 June 2007 (EDT)''': Using this protocol (including the measurement of competence), I obtained efficiencies of 3E8 for TOP10 cells.		*'''[[User:Reshma P. Shetty\|Reshma]] 11:36, 12 June 2007 (EDT)''': Using this protocol (including the measurement of competence), I obtained efficiencies of 3E8 for TOP10 cells.
		+	--[[User:Andy Lane\|Andy Lane]] 16:33, 26 November 2010 (EST) I achieved competencies of 8E7 for DH5a cells and 5.5E7 for XL-10 Gold cells. DH5a was harvested at OD600 of 0.3; XL-10 Gold at 0.35) However, I lost many cells during the centrifugation steps and froze down at a lower density than recommended. The protocol states that a 1:5 dilution in SOC should have an OD600 of 1-1.5 at freezing; mine was only 0.55 - 0.6 for both strains.

	==Questions==		==Questions==
	*'''[[User:Bcanton\|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?		*'''[[User:Bcanton\|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?

Andy Lane: /* Comments */

2010-11-25T17:31:33Z

Comments

←Older revision		Revision as of 17:31, 25 November 2010
Line 13:		Line 13:

	*'''[[User:Reshma P. Shetty\|Reshma]] 15:06, 5 June 2007 (EDT)''': Growth of the overnight 250mL culture to OD600nm of 0.3 from seed stocks took exactly 17 hours at 20°C.		*'''[[User:Reshma P. Shetty\|Reshma]] 15:06, 5 June 2007 (EDT)''': Growth of the overnight 250mL culture to OD600nm of 0.3 from seed stocks took exactly 17 hours at 20°C.
		+
		+	* '''[[User:Andy Lane\|Andy Lane]] 12:31, 25 November 2010 (EST)''': Doubling time for DH5a or XL-10 Gold @ 20˚C in SOB media seems to be approx 2h 30m to 3h in my hands. This may help in planning the timing of the protocol.

	==Transformation efficiencies==		==Transformation efficiencies==

Reshma P. Shetty at 15:36, 12 June 2007

2007-06-12T15:36:47Z

←Older revision		Revision as of 15:36, 12 June 2007
Line 13:		Line 13:

	*'''[[User:Reshma P. Shetty\|Reshma]] 15:06, 5 June 2007 (EDT)''': Growth of the overnight 250mL culture to OD600nm of 0.3 from seed stocks took exactly 17 hours at 20°C.		*'''[[User:Reshma P. Shetty\|Reshma]] 15:06, 5 June 2007 (EDT)''': Growth of the overnight 250mL culture to OD600nm of 0.3 from seed stocks took exactly 17 hours at 20°C.
		+
		+	==Transformation efficiencies==
		+	*'''[[User:Reshma P. Shetty\|Reshma]] 11:36, 12 June 2007 (EDT)''': Using this protocol (including the measurement of competence), I obtained efficiencies of 3E8 for TOP10 cells.

	==Questions==		==Questions==
	*'''[[User:Bcanton\|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?		*'''[[User:Bcanton\|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?

Reshma P. Shetty at 19:07, 5 June 2007

2007-06-05T19:07:34Z

←Older revision		Revision as of 19:07, 5 June 2007
Line 1:		Line 1:
-	'''[[User:Bcanton\|BC]] 11:22, 23 August 2006 (EDT)''': I used this protocol to make BL21(DE3) competent. I obtained these transformation efficiencies -	+	==Comments==
-	*1.50E6 when transforming .01ng of prepped pUC18 DNA (1 colony on plate, so not very accurate number)	+	*'''[[User:Bcanton\|BC]] 11:22, 23 August 2006 (EDT)''': I used this protocol to make BL21(DE3) competent. I obtained these transformation efficiencies -
-	*2.25E5 when transforming 1.0ng of prepped pUC18 DNA (15 colonies on plate)	+	**1.50E6 when transforming .01ng of prepped pUC18 DNA (1 colony on plate, so not very accurate number)
		+	**2.25E5 when transforming 1.0ng of prepped pUC18 DNA (15 colonies on plate)

	The second figure is more likely to be accurate as I got a very small number of colonies when transforming .01ng of DNA.		The second figure is more likely to be accurate as I got a very small number of colonies when transforming .01ng of DNA.
Line 10:		Line 11:
	*I incubated the transformed cells in [[2XYT]] instead of [[SOC]].		*I incubated the transformed cells in [[2XYT]] instead of [[SOC]].
	*Time constraints meant that I only incubated the transformed cells for 30mins prior to plating.		*Time constraints meant that I only incubated the transformed cells for 30mins prior to plating.
-
-	~~'''[[User:Bcanton\|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?~~

	*'''[[User:Reshma P. Shetty\|Reshma]] 15:06, 5 June 2007 (EDT)''': Growth of the overnight 250mL culture to OD600nm of 0.3 from seed stocks took exactly 17 hours at 20°C.		*'''[[User:Reshma P. Shetty\|Reshma]] 15:06, 5 June 2007 (EDT)''': Growth of the overnight 250mL culture to OD600nm of 0.3 from seed stocks took exactly 17 hours at 20°C.
		+
		+	==Questions==
		+	*'''[[User:Bcanton\|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?

Reshma P. Shetty at 19:06, 5 June 2007

2007-06-05T19:06:44Z

←Older revision		Revision as of 19:06, 5 June 2007
Line 12:		Line 12:

	'''[[User:Bcanton\|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?		'''[[User:Bcanton\|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?
		+
		+	*'''[[User:Reshma P. Shetty\|Reshma]] 15:06, 5 June 2007 (EDT)''': Growth of the overnight 250mL culture to OD600nm of 0.3 from seed stocks took exactly 17 hours at 20°C.

Barry Canton at 16:07, 6 February 2007

2007-02-06T16:07:55Z

←Older revision		Revision as of 16:07, 6 February 2007
Line 10:		Line 10:
	*I incubated the transformed cells in [[2XYT]] instead of [[SOC]].		*I incubated the transformed cells in [[2XYT]] instead of [[SOC]].
	*Time constraints meant that I only incubated the transformed cells for 30mins prior to plating.		*Time constraints meant that I only incubated the transformed cells for 30mins prior to plating.
		+
		+	'''[[User:Bcanton\|BC]] 11:07, 6 February 2007 (EST):''' What OD should the seed cultures be at the time glycerol is added?