Lory:Quick transformant screen - Revision history

Bentley Lim: /* Notes */

2009-05-10T01:11:36Z

Notes

←Older revision		Revision as of 01:11, 10 May 2009
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	'''*[[User:Khturner\|Khturner]] 16:51, 15 April 2007 (EDT)''': You know, you're probably right. It used to help me when I had problems cloning and a successful construct was rare, but now it's become pretty routine, and the screening does only take a day by colony PCR. I'll happily remove this from the protocols page, thanks for the comments.		'''*[[User:Khturner\|Khturner]] 16:51, 15 April 2007 (EDT)''': You know, you're probably right. It used to help me when I had problems cloning and a successful construct was rare, but now it's become pretty routine, and the screening does only take a day by colony PCR. I'll happily remove this from the protocols page, thanks for the comments.
		+
		+	'''*[[User:Blim\|Bentley Lim]] May 9, 2009''': Don't take down this protocol. While many basic cloning experiments use sequenced and up-to-date vectors from companies, etc., many plasmids that still exist have no NCBI entry. Sometimes, it's impossible to reconstruct the plasmid yourself from papers published before 1990. Therefore, with some cloning experiments, you just need a quick-and-dirty way to see if an insert went in and then you can sequence out to carry out the rest of your cloning procedures.

	==Contact==		==Contact==

Reshma P. Shetty at 01:32, 10 May 2007

2007-05-10T01:32:42Z

←Older revision		Revision as of 01:32, 10 May 2007
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	*[[User:Khturner]]		*[[User:Khturner]]

-	[[Category:Protocol]]	+	[[Category:Protocol]] [[Category:Escherichia coli]] [[Category:In vitro]] [[Category:DNA]]

	''Last edited December 7, 2006''		''Last edited December 7, 2006''

Khturner: /* Notes */

2007-04-15T20:51:46Z

Notes

←Older revision		Revision as of 20:51, 15 April 2007
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	In this example, my vector is 5.5 kb, and the insert is ~1.8 kb. The negative control colonies are recircularized vector, and run at the right size. The lane marked X is a failure. It contains the recircularized vector that we're not interested in, and while I'm not entirely sure what that strong band is, it's not what we're looking for either. The final lane, marked +, is correct, a single band running at around 7.5 kb.		In this example, my vector is 5.5 kb, and the insert is ~1.8 kb. The negative control colonies are recircularized vector, and run at the right size. The lane marked X is a failure. It contains the recircularized vector that we're not interested in, and while I'm not entirely sure what that strong band is, it's not what we're looking for either. The final lane, marked +, is correct, a single band running at around 7.5 kb.
	==Notes==		==Notes==
-	'''*[[User:Khturner\|Khturner]] 10:19, 7 December 2006 (EST)''': The first couple of times you try this, it might be tough to get a feel for the viscosity of the sample at each stage, but once you get the method to work, it pretty much eliminates the need to optimize your ligation and transformation,	+	'''*[[User:Khturner\|Khturner]] 10:19, 7 December 2006 (EST)''': The first couple of times you try this, it might be tough to get a feel for the viscosity of the sample at each stage, but once you get the method to work, it pretty much eliminates the need to optimize your ligation and transformation.

	'''[[User:Smoore\|Sean Moore]] Feb. 5, 2007'''		'''[[User:Smoore\|Sean Moore]] Feb. 5, 2007'''
	Is this necessary? This adds a whole day to the screening procedure? You could have done PCR on the colonies in a few hours from the original plate if you needed to screen for colonies with the correct insert. I don't see more colonies on the "vector + insert" plate than the "vector only" plate, I usually don't even bother screening and assume something is wrong with the fragments. I have never "optimized" ligations. Also, some replication origins cause significant catenation so analysis in the supercoiled form is impractical/unreliable.		Is this necessary? This adds a whole day to the screening procedure? You could have done PCR on the colonies in a few hours from the original plate if you needed to screen for colonies with the correct insert. I don't see more colonies on the "vector + insert" plate than the "vector only" plate, I usually don't even bother screening and assume something is wrong with the fragments. I have never "optimized" ligations. Also, some replication origins cause significant catenation so analysis in the supercoiled form is impractical/unreliable.
		+
		+	'''*[[User:Khturner\|Khturner]] 16:51, 15 April 2007 (EDT)''': You know, you're probably right. It used to help me when I had problems cloning and a successful construct was rare, but now it's become pretty routine, and the screening does only take a day by colony PCR. I'll happily remove this from the protocols page, thanks for the comments.

	==Contact==		==Contact==

Smoore: /* Notes */

2007-02-05T19:34:30Z

Notes

←Older revision		Revision as of 19:34, 5 February 2007
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	'''[[User:Smoore\|Sean Moore]] Feb. 5, 2007'''		'''[[User:Smoore\|Sean Moore]] Feb. 5, 2007'''
-	Is this necessary? This adds a whole day to the screening procedure? You could have done PCR on the colonies in a few hours from the original plate if you needed to screen for colonies with the correct insert. I don't see more colonies on the "vector + insert" plate than the "vector only" plate, I usually don't even bother screening ~~ans~~ assume something is wrong with the fragments. I have never "optimized" ligations. Also, some replication origins cause significant catenation so analysis in the supercoiled form is impractical/unreliable.	+	Is this necessary? This adds a whole day to the screening procedure? You could have done PCR on the colonies in a few hours from the original plate if you needed to screen for colonies with the correct insert. I don't see more colonies on the "vector + insert" plate than the "vector only" plate, I usually don't even bother screening and assume something is wrong with the fragments. I have never "optimized" ligations. Also, some replication origins cause significant catenation so analysis in the supercoiled form is impractical/unreliable.

	==Contact==		==Contact==

Smoore: /* Notes */

2007-02-05T19:33:47Z

Notes

←Older revision		Revision as of 19:33, 5 February 2007
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	'''[[User:Smoore\|Sean Moore]] Feb. 5, 2007'''		'''[[User:Smoore\|Sean Moore]] Feb. 5, 2007'''
-	Is this necessary? This adds a whole day to the screening procedure? You could have done PCR on the colonies in a few hours from the original plate if you needed to screen for colonies with the correct insert. II don't see more colonies on the "vector + insert" plate than the "vector only" plate, I usually don't even bother screening. I have never "optimized" ligations. Also, some replication origins cause significant catenation so analysis in the supercoiled form is impractical/unreliable.	+	Is this necessary? This adds a whole day to the screening procedure? You could have done PCR on the colonies in a few hours from the original plate if you needed to screen for colonies with the correct insert. I don't see more colonies on the "vector + insert" plate than the "vector only" plate, I usually don't even bother screening ans assume something is wrong with the fragments. I have never "optimized" ligations. Also, some replication origins cause significant catenation so analysis in the supercoiled form is impractical/unreliable.

	==Contact==		==Contact==

Smoore: /* Notes */

2007-02-05T19:33:08Z

Notes

←Older revision		Revision as of 19:33, 5 February 2007
Line 32:		Line 32:
	'''*[[User:Khturner\|Khturner]] 10:19, 7 December 2006 (EST)''': The first couple of times you try this, it might be tough to get a feel for the viscosity of the sample at each stage, but once you get the method to work, it pretty much eliminates the need to optimize your ligation and transformation,		'''*[[User:Khturner\|Khturner]] 10:19, 7 December 2006 (EST)''': The first couple of times you try this, it might be tough to get a feel for the viscosity of the sample at each stage, but once you get the method to work, it pretty much eliminates the need to optimize your ligation and transformation,

-	'''*[[~~smoore~~]] Feb. 5, 2007'''	+	'''[[User:Smoore\|Sean Moore]] Feb. 5, 2007'''
	Is this necessary? This adds a whole day to the screening procedure? You could have done PCR on the colonies in a few hours from the original plate if you needed to screen for colonies with the correct insert. II don't see more colonies on the "vector + insert" plate than the "vector only" plate, I usually don't even bother screening. I have never "optimized" ligations. Also, some replication origins cause significant catenation so analysis in the supercoiled form is impractical/unreliable.		Is this necessary? This adds a whole day to the screening procedure? You could have done PCR on the colonies in a few hours from the original plate if you needed to screen for colonies with the correct insert. II don't see more colonies on the "vector + insert" plate than the "vector only" plate, I usually don't even bother screening. I have never "optimized" ligations. Also, some replication origins cause significant catenation so analysis in the supercoiled form is impractical/unreliable.

Smoore: /* Notes */

2007-02-05T19:31:16Z

Notes

←Older revision		Revision as of 19:31, 5 February 2007
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	==Notes==		==Notes==
	'''*[[User:Khturner\|Khturner]] 10:19, 7 December 2006 (EST)''': The first couple of times you try this, it might be tough to get a feel for the viscosity of the sample at each stage, but once you get the method to work, it pretty much eliminates the need to optimize your ligation and transformation,		'''*[[User:Khturner\|Khturner]] 10:19, 7 December 2006 (EST)''': The first couple of times you try this, it might be tough to get a feel for the viscosity of the sample at each stage, but once you get the method to work, it pretty much eliminates the need to optimize your ligation and transformation,
		+
		+	'''*[[smoore]] Feb. 5, 2007'''
		+	Is this necessary? This adds a whole day to the screening procedure? You could have done PCR on the colonies in a few hours from the original plate if you needed to screen for colonies with the correct insert. II don't see more colonies on the "vector + insert" plate than the "vector only" plate, I usually don't even bother screening. I have never "optimized" ligations. Also, some replication origins cause significant catenation so analysis in the supercoiled form is impractical/unreliable.

	==Contact==		==Contact==

Khturner: Turner:Quick Transformant Screen moved to Lory:Quick transformant screen: Conforming with Help:Page naming conventions

2006-12-09T15:39:22Z

Turner:Quick Transformant Screen moved to Lory:Quick transformant screen: Conforming with Help:Page naming conventions

←Older revision

Revision as of 15:39, 9 December 2006

Khturner at 17:23, 7 December 2006

2006-12-07T17:23:30Z

←Older revision		Revision as of 17:23, 7 December 2006
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	==Overview==		==Overview==
-	This is an easy method for screening a large amount of transformants in a cloning procedure, relieving you of the need to worry about optimizing your ligation or doing a huge amount of minipreps. Works best if a vector-only control ligation is transformed as well.	+	This is an easy method for screening a large amount of [[Bacterial_Transformation\|transformants]] in a cloning procedure, relieving you of the need to worry about optimizing your [[DNA_ligation\|ligation]] or doing a huge amount of [[miniprep\|minipreps]]. Works best if a vector-only control ligation is transformed as well.

	==Materials==		==Materials==

Khturner at 15:19, 7 December 2006

2006-12-07T15:19:29Z

←Older revision		Revision as of 15:19, 7 December 2006
Line 1:		Line 1:
	==Overview==		==Overview==
-	This is an easy method for screening a large amount of transformants in a cloning procedure, relieving you of the need to worry about optimizing your ligation or doing a huge amount of minipreps. Works best if a vector-only control is transformed as well.	+	This is an easy method for screening a large amount of transformants in a cloning procedure, relieving you of the need to worry about optimizing your ligation or doing a huge amount of minipreps. Works best if a vector-only control ligation is transformed as well.

	==Materials==		==Materials==