Studier Lysate Prep - Revision history

Torsten Waldminghaus at 17:22, 23 February 2009

2009-02-23T17:22:29Z

←Older revision		Revision as of 17:22, 23 February 2009
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		+	{{back to protocols}}
	== Summary ==		== Summary ==
	How to make a lysate from a plaque preparation. We also use this protocol for preparation of a quick stock from previously made lysate prep.		How to make a lysate from a plaque preparation. We also use this protocol for preparation of a quick stock from previously made lysate prep.

Reshma P. Shetty at 20:17, 8 May 2007

2007-05-08T20:17:23Z

←Older revision		Revision as of 20:17, 8 May 2007
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	How to make a lysate from a plaque preparation. We also use this protocol for preparation of a quick stock from previously made lysate prep.		How to make a lysate from a plaque preparation. We also use this protocol for preparation of a quick stock from previously made lysate prep.

-	== Protocol ==	+	==Protocol==
	# Add 5mL fresh overnight (BL21 if wild-type T7) into 15 mL of [[T broth]] (125 mL flask).		# Add 5mL fresh overnight (BL21 if wild-type T7) into 15 mL of [[T broth]] (125 mL flask).
	#* I often use [[LB]] --[[User:Skosuri\|Sri Kosuri]] 15:18, 3 Jun 2005 (EDT)		#* I often use [[LB]] --[[User:Skosuri\|Sri Kosuri]] 15:18, 3 Jun 2005 (EDT)
Line 13:		Line 13:
	# Store tube in fridge away from light (usually good indefinitely, e.g., retains absorption well).		# Store tube in fridge away from light (usually good indefinitely, e.g., retains absorption well).

-	== References ==	+	==References==
-	Studier~~, FW~~ Virology ~~'''39''':562 (~~1969~~) Link~~: [~~http~~:~~//www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=4902069&query_hl=1 Pubmed~~]	+	<biblio>
		+	#Studier-Virology-1969 pmid=4902069
		+	</biblio>
		+
		+	[[Category:Protocol]] [[Category:T7]]

Skosuri: /* Protocol */

2005-09-22T17:44:38Z

Protocol

←Older revision		Revision as of 17:44, 22 September 2005
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	#* I often use [[LB]] --[[User:Skosuri\|Sri Kosuri]] 15:18, 3 Jun 2005 (EDT)		#* I often use [[LB]] --[[User:Skosuri\|Sri Kosuri]] 15:18, 3 Jun 2005 (EDT)
	# Add a single plaque to above (or a drop of lysate)		# Add a single plaque to above (or a drop of lysate)
-	# Shake at 30°C until lysis is visually apparent	+	# Shake at 30°C until lysis is visually apparent (2-3hr)
	# '''As soon as lysis is observed''' and appears to be complete, add 1g of NaCL to the flask and dissolve by shaking.		# '''As soon as lysis is observed''' and appears to be complete, add 1g of NaCL to the flask and dissolve by shaking.
	#* Adding NaCl prevents phage particles from getting pulled down during centrifugation.		#* Adding NaCl prevents phage particles from getting pulled down during centrifugation.

Skosuri: /* Protocol */

2005-06-03T20:16:14Z

Protocol

←Older revision		Revision as of 20:16, 3 June 2005
Line 5:		Line 5:
	# Add 5mL fresh overnight (BL21 if wild-type T7) into 15 mL of [[T broth]] (125 mL flask).		# Add 5mL fresh overnight (BL21 if wild-type T7) into 15 mL of [[T broth]] (125 mL flask).
	#* I often use [[LB]] --[[User:Skosuri\|Sri Kosuri]] 15:18, 3 Jun 2005 (EDT)		#* I often use [[LB]] --[[User:Skosuri\|Sri Kosuri]] 15:18, 3 Jun 2005 (EDT)
-	# Add a single plaque to above	+	# Add a single plaque to above (or a drop of lysate)
	# Shake at 30°C until lysis is visually apparent		# Shake at 30°C until lysis is visually apparent
	# '''As soon as lysis is observed''' and appears to be complete, add 1g of NaCL to the flask and dissolve by shaking.		# '''As soon as lysis is observed''' and appears to be complete, add 1g of NaCL to the flask and dissolve by shaking.

Skosuri: /* Summary */

2005-06-03T19:35:04Z

Summary

←Older revision		Revision as of 19:35, 3 June 2005
Line 1:		Line 1:
	== Summary ==		== Summary ==
-	How to make a lysate from a plaque preparation.	+	How to make a lysate from a plaque preparation. We also use this protocol for preparation of a quick stock from previously made lysate prep.
		+
	== Protocol ==		== Protocol ==
	# Add 5mL fresh overnight (BL21 if wild-type T7) into 15 mL of [[T broth]] (125 mL flask).		# Add 5mL fresh overnight (BL21 if wild-type T7) into 15 mL of [[T broth]] (125 mL flask).

Skosuri at 19:19, 3 June 2005

2005-06-03T19:19:28Z

←Older revision		Revision as of 19:19, 3 June 2005
Line 2:		Line 2:
	How to make a lysate from a plaque preparation.		How to make a lysate from a plaque preparation.
	== Protocol ==		== Protocol ==
-	# Add 5mL fresh overnight (BL21 if wild-type T7) into 15 mL of T broth (125 mL flask).	+	# Add 5mL fresh overnight (BL21 if wild-type T7) into 15 mL of [[T broth]] (125 mL flask).
-	#* I often use LB --[[User:Skosuri\|Sri Kosuri]] 15:18, 3 Jun 2005 (EDT)	+	#* I often use [[LB]] --[[User:Skosuri\|Sri Kosuri]] 15:18, 3 Jun 2005 (EDT)
	# Add a single plaque to above		# Add a single plaque to above
	# Shake at 30°C until lysis is visually apparent		# Shake at 30°C until lysis is visually apparent

Skosuri at 19:18, 3 June 2005

2005-06-03T19:18:37Z

←Older revision		Revision as of 19:18, 3 June 2005
Line 2:		Line 2:
	How to make a lysate from a plaque preparation.		How to make a lysate from a plaque preparation.
	== Protocol ==		== Protocol ==
-	# Add 5mL fresh overnight (BL21 if wild-type T7) into 15 mL of T broth (125 mL flask)	+	# Add 5mL fresh overnight (BL21 if wild-type T7) into 15 mL of T broth (125 mL flask).
		+	#* I often use LB --[[User:Skosuri\|Sri Kosuri]] 15:18, 3 Jun 2005 (EDT)
	# Add a single plaque to above		# Add a single plaque to above
	# Shake at 30°C until lysis is visually apparent		# Shake at 30°C until lysis is visually apparent
	# '''As soon as lysis is observed''' and appears to be complete, add 1g of NaCL to the flask and dissolve by shaking.		# '''As soon as lysis is observed''' and appears to be complete, add 1g of NaCL to the flask and dissolve by shaking.
-	#* Studier has observed that leaving released phage in the cell debris causes a reduction in the kinetics of phage binding (personal communication). It becomes difficult to get good time courses in this case. It is recommended when making a large stock that will be used in timing experiments, that you check that most of your phage adsorb to the cell in the first 30 seconds.	+	#* Adding NaCl prevents phage particles from getting pulled down during centrifugation.
-	#	+	# Once NaCl is dissolved, centrifuge for 10 min at 10,000 rpm at 4°C.
		+	#* Studier has observed that leaving released phage in the cell debris causes a reduction in the kinetics of phage binding (personal communication). It becomes difficult to get good time courses in this case. This can often happen even over the course of an hour. So it is important to catch lysis and spinning down the debris as soon as possible. It is recommended when making a large stock that will be used in timing experiments, that you check that most of your phage adsorb to the cell in the first 30 seconds.
		+	# Store tube in fridge away from light (usually good indefinitely, e.g., retains absorption well).
		+
	== References ==		== References ==
	Studier, FW Virology '''39''':562 (1969) Link: [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=4902069&query_hl=1 Pubmed]		Studier, FW Virology '''39''':562 (1969) Link: [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=4902069&query_hl=1 Pubmed]

Skosuri at 19:13, 3 June 2005

2005-06-03T19:13:29Z

New page

== Summary ==
How to make a lysate from a plaque preparation.
== Protocol ==
# Add 5mL fresh overnight (BL21 if wild-type T7) into 15 mL of T broth (125 mL flask)
# Add a single plaque to above
# Shake at 30°C until lysis is visually apparent
# '''As soon as lysis is observed''' and appears to be complete, add 1g of NaCL to the flask and dissolve by shaking.
#* Studier has observed that leaving released phage in the cell debris causes a reduction in the kinetics of phage binding (personal communication). It becomes difficult to get good time courses in this case. It is recommended when making a large stock that will be used in timing experiments, that you check that most of your phage adsorb to the cell in the first 30 seconds.
#
== References ==
Studier, FW Virology '''39''':562 (1969) Link: [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=4902069&query_hl=1 Pubmed]