OriC/ter ratio determination - Revision history

Torsten Waldminghaus at 07:58, 9 February 2010

2010-02-09T07:58:44Z

←Older revision		Revision as of 07:58, 9 February 2010
Line 5:		Line 5:

	==Abstract==		==Abstract==
-	This protocol describes how one can determine the ~~ration~~ of the origin of ~~termination~~ ''oriC'' ~~and~~ the terminus of replication ''ter'' in ''Escherichia coli''. That is for example important for [[cell cycle analysis]]. To get the ratio one could use Southern Blot and quantification of the bands. However, this protocol describes the use of qPCR which is a bit faster.	+	This protocol describes how one can determine the ratio of the origin of replication, ''oriC'', to the terminus of replication, ''ter'', in ''Escherichia coli''. That is for example important for [[cell cycle analysis]]. To get the ratio one could use Southern Blot and quantification of the bands. However, this protocol describes the use of qPCR which is a bit faster.

	==Procedure==		==Procedure==
	*First you need to purify the DNA you want to analyse (see for example [[Chromosomal DNA isolation from E. coli]]).		*First you need to purify the DNA you want to analyse (see for example [[Chromosomal DNA isolation from E. coli]]).
-	*To normalize the data for your sample you need chromosomal DNA from cells that are not replicating and have only complete chromosomes. ~~For this there are two possibilities. First, you can use E. coli cells in the late stationary phase. Second, you could use~~ Rifampicin treated cells. Rif stops replication initiation. The ongoing replications are finished and you end up with whole chromosomes after a while. How to get Rif cells you can find here: [[User:Torsten Waldminghaus/flow cytometry notes \|flow cytometry notes]]. It would be ideal to check your cells by flow cytometry (if they have no ongoing replication).	+	*To normalize the data for your sample you need chromosomal DNA from cells that are not replicating and have only complete chromosomes. Rifampicin treated cells are suitable since Rif stops replication initiation. The ongoing replications are finished and you end up with whole chromosomes after a while. How to get Rif cells you can find here: [[User:Torsten Waldminghaus/flow cytometry notes \|flow cytometry notes]]. It would be ideal to check your cells by flow cytometry (if they have no ongoing replication).
	*Digest 100ng of your DNA (sample and control) in 10μL volume with EcoRI for 30min at 37°C. '''Note:''' This is to make the following qPCR more accurate since PCR of entire chromosomes might give some unwanted bias.		*Digest 100ng of your DNA (sample and control) in 10μL volume with EcoRI for 30min at 37°C. '''Note:''' This is to make the following qPCR more accurate since PCR of entire chromosomes might give some unwanted bias.
	*add 90μL dH<sub>2</sub>O		*add 90μL dH<sub>2</sub>O

Torsten Waldminghaus at 10:46, 2 March 2009

2009-03-02T10:46:08Z

←Older revision		Revision as of 10:46, 2 March 2009
Line 21:		Line 21:
	!for 250μL		!for 250μL
	\|-		\|-
-	\|22.5μL primer fw	+	\|22.5μL primer fw (100pmol/μL)
	\|-		\|-
-	\|22.5μL primer rv	+	\|22.5μL primer rv (100pmol/μL)
	\|-		\|-
-	\|6.25μL probe	+	\|6.25μL probe (100pmol/μL)
	\|-		\|-
	\|198.75μL ddH<sub>2</sub>O		\|198.75μL ddH<sub>2</sub>O

Torsten Waldminghaus at 10:18, 27 February 2009

2009-02-27T10:18:31Z

←Older revision		Revision as of 10:18, 27 February 2009
Line 6:		Line 6:
	==Abstract==		==Abstract==
	This protocol describes how one can determine the ration of the origin of termination ''oriC'' and the terminus of replication ''ter'' in ''Escherichia coli''. That is for example important for [[cell cycle analysis]]. To get the ratio one could use Southern Blot and quantification of the bands. However, this protocol describes the use of qPCR which is a bit faster.		This protocol describes how one can determine the ration of the origin of termination ''oriC'' and the terminus of replication ''ter'' in ''Escherichia coli''. That is for example important for [[cell cycle analysis]]. To get the ratio one could use Southern Blot and quantification of the bands. However, this protocol describes the use of qPCR which is a bit faster.
-
-	~~==Materials==~~
-	List everything necessary to perform the protocol here. Include all information about suppliers, ordering details, etc. Links to the suppliers' page on that material are also appropriate and encouraged. Please be aware that users of this protocol may not be working in the same country as you.
-	~~===Reagents===~~
-	~~Biological resources e.g. cell lines, buffers (link to a method for making them), enzymes, chemicals, kits, etc.~~
-	~~===Equipment===~~
-	~~Any equipment used to perform the protocol (link to a method for using them).~~

	==Procedure==		==Procedure==
Line 45:		Line 38:
	*run qPCR		*run qPCR
	*calculate oriC/ter ratio normalized to the oriC/ter ratio of rif sample		*calculate oriC/ter ratio normalized to the oriC/ter ratio of rif sample
-
-	~~==Critical steps==~~
-	~~Referenced from the main protocol, a more thorough explanation of particularly important steps in the protocol.~~
-
-
-
-	~~==Notes==~~
-	Referenced from the main protocol, any comments about the protocol should be made here; i.e. how it was developed. Any comments added should be signed (by adding <nowiki>*'''~~~~''':</nowiki> in front) and explained. Links to FAQs/tips provided by other sources such as the manufacturer or other websites would be best made here.<br>
-	~~Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.<br>~~


	==Discussion==		==Discussion==
-	You can [[Talk:{{PAGENAME}}\|discuss this protocol]].	+	You can [[Talk:{{PAGENAME}}\|discuss this protocol]].
-		+
-	~~Tag this page with categories to allow easier indexing and searching. See [[Categories]] for information on existing categories~~.	+

Torsten Waldminghaus: /* Procedure */

2009-02-27T10:17:37Z

Procedure

←Older revision		Revision as of 10:17, 27 February 2009
Line 21:		Line 21:
	*use 10μL as template for 1 PCR reaction		*use 10μL as template for 1 PCR reaction
	*for the qPCR use primers ter (for terminus) and 3921366 (for oriC). Details are here: [[User:Torsten_Waldminghaus/Primers]].		*for the qPCR use primers ter (for terminus) and 3921366 (for oriC). Details are here: [[User:Torsten_Waldminghaus/Primers]].
		+	*Make primer mixes for each set of primers:
		+
		+
	{\|border="1"		{\|border="1"
	\|+ '''Primer Mix'''		\|+ '''Primer Mix'''
Line 34:		Line 37:
	\|}		\|}

-		+	*For each each primer set make a master mix corresponding to three parallels per primer set per DNA (includeing one or two extra for pipetting errors)
-	~~If you find it helpfull you could use the following icons~~ to ~~highlight important parts:~~	+	#12.5μL TaqMan Mix
-		+	#2.5μL primer mix (see table above)
-	~~[[Image:Difficult step~~.~~png]] icon to highlight difficult steps~~	+	*pipett 15μL in wells on 96-well PCR plate
-		+	*add 10μL DNA
-	~~[[Image:Critical step~~.~~png]] warn about critical steps~~	+	*spin down plate at 1000 rpm to collect sample on the bottom
-		+	*run qPCR
-	~~[[Image:Time required.png]] help people plan how long steps will take~~	+	*calculate oriC/ter ratio normalized to the oriC/ter ratio of rif sample
-		+
-	~~[[Image:Pause point.png]] where protocol can be interrupted~~	+
-		+
-	~~[[Image:Optional step.png]] steps that can be omitted or included~~	+

	==Critical steps==		==Critical steps==

Torsten Waldminghaus at 08:55, 27 February 2009

2009-02-27T08:55:07Z

←Older revision		Revision as of 08:55, 27 February 2009
Line 20:		Line 20:
	*add 90μL dH<sub>2</sub>O		*add 90μL dH<sub>2</sub>O
	*use 10μL as template for 1 PCR reaction		*use 10μL as template for 1 PCR reaction
		+	*for the qPCR use primers ter (for terminus) and 3921366 (for oriC). Details are here: [[User:Torsten_Waldminghaus/Primers]].
	{\|border="1"		{\|border="1"
-	\|+ '''Primer ~~Mixe~~'''	+	\|+ '''Primer Mix'''
	!for 250μL		!for 250μL
	\|-		\|-

Torsten Waldminghaus: /* Procedure */

2009-02-27T08:49:36Z

Procedure

←Older revision		Revision as of 08:49, 27 February 2009
Line 15:		Line 15:

	==Procedure==		==Procedure==
-	~~A step~~ by ~~step guide~~ to the ~~experimental procedure~~.	+	*First you need to purify the DNA you want to analyse (see for example [[Chromosomal DNA isolation from E. coli]]).
		+	*To normalize the data for your sample you need chromosomal DNA from cells that are not replicating and have only complete chromosomes. For this there are two possibilities. First, you can use E. coli cells in the late stationary phase. Second, you could use Rifampicin treated cells. Rif stops replication initiation. The ongoing replications are finished and you end up with whole chromosomes after a while. How to get Rif cells you can find here: [[User:Torsten Waldminghaus/flow cytometry notes \|flow cytometry notes]]. It would be ideal to check your cells by flow cytometry (if they have no ongoing replication).
		+	*Digest 100ng of your DNA (sample and control) in 10μL volume with EcoRI for 30min at 37°C. '''Note:''' This is to make the following qPCR more accurate since PCR of entire chromosomes might give some unwanted bias.
		+	*add 90μL dH<sub>2</sub>O
		+	*use 10μL as template for 1 PCR reaction
		+	{\|border="1"
		+	\|+ '''Primer Mixe'''
		+	!for 250μL
		+	\|-
		+	\|22.5μL primer fw
		+	\|-
		+	\|22.5μL primer rv
		+	\|-
		+	\|6.25μL probe
		+	\|-
		+	\|198.75μL ddH<sub>2</sub>O
		+	\|}
		+

	If you find it helpfull you could use the following icons to highlight important parts:		If you find it helpfull you could use the following icons to highlight important parts:

Torsten Waldminghaus: /* Abstract */

2009-02-27T08:27:55Z

Abstract

←Older revision		Revision as of 08:27, 27 February 2009
Line 5:		Line 5:

	==Abstract==		==Abstract==
-	This protocol describes how one can determine the ration of the origin of termination ''oriC'' and the terminus of replication ''ter'' in ''Escherichia coli''. That is for example important for [[cell cycle analysis]].	+	This protocol describes how one can determine the ration of the origin of termination ''oriC'' and the terminus of replication ''ter'' in ''Escherichia coli''. That is for example important for [[cell cycle analysis]]. To get the ratio one could use Southern Blot and quantification of the bands. However, this protocol describes the use of qPCR which is a bit faster.

	==Materials==		==Materials==

Torsten Waldminghaus at 16:31, 26 February 2009

2009-02-26T16:31:45Z

←Older revision		Revision as of 16:31, 26 February 2009
Line 1:		Line 1:
		+	==Curators==
		+	'''[[User:Torsten Waldminghaus \| Torsten Waldminghaus]]'''
		+
		+	''Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute. The OpenWetWare community is currently [[OpenWetWare:Information management/Protocol curators\|discussing the idea of protocol curators]]. Please contribute.''
		+
		+	==Abstract==
	This protocol describes how one can determine the ration of the origin of termination ''oriC'' and the terminus of replication ''ter'' in ''Escherichia coli''. That is for example important for [[cell cycle analysis]].		This protocol describes how one can determine the ration of the origin of termination ''oriC'' and the terminus of replication ''ter'' in ''Escherichia coli''. That is for example important for [[cell cycle analysis]].
		+
		+	==Materials==
		+	List everything necessary to perform the protocol here. Include all information about suppliers, ordering details, etc. Links to the suppliers' page on that material are also appropriate and encouraged. Please be aware that users of this protocol may not be working in the same country as you.
		+	===Reagents===
		+	Biological resources e.g. cell lines, buffers (link to a method for making them), enzymes, chemicals, kits, etc.
		+	===Equipment===
		+	Any equipment used to perform the protocol (link to a method for using them).
		+
		+	==Procedure==
		+	A step by step guide to the experimental procedure.
		+
		+	If you find it helpfull you could use the following icons to highlight important parts:
		+
		+	[[Image:Difficult step.png]] icon to highlight difficult steps
		+
		+	[[Image:Critical step.png]] warn about critical steps
		+
		+	[[Image:Time required.png]] help people plan how long steps will take
		+
		+	[[Image:Pause point.png]] where protocol can be interrupted
		+
		+	[[Image:Optional step.png]] steps that can be omitted or included
		+
		+	==Critical steps==
		+	Referenced from the main protocol, a more thorough explanation of particularly important steps in the protocol.
		+
		+
		+
		+	==Notes==
		+	Referenced from the main protocol, any comments about the protocol should be made here; i.e. how it was developed. Any comments added should be signed (by adding <nowiki>*'''~~~~''':</nowiki> in front) and explained. Links to FAQs/tips provided by other sources such as the manufacturer or other websites would be best made here.<br>
		+	Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.<br>
		+
		+
		+	==Discussion==
		+	You can [[Talk:{{PAGENAME}}\|discuss this protocol]].
		+
		+	Tag this page with categories to allow easier indexing and searching. See [[Categories]] for information on existing categories.

Torsten Waldminghaus: New page: This protocol describes how one can determine the ration of the origin of termination ''oriC'' and the terminus of replication ''ter'' in ''Escherichia coli''. That is for example importan...

2009-02-26T16:25:51Z

New page: This protocol describes how one can determine the ration of the origin of termination ''oriC'' and the terminus of replication ''ter'' in ''Escherichia coli''. That is for example importan...

New page

This protocol describes how one can determine the ration of the origin of termination ''oriC'' and the terminus of replication ''ter'' in ''Escherichia coli''. That is for example important for [[cell cycle analysis]].