OriC/ter ratio determination

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==Abstract==
==Abstract==
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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]].
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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.
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==Materials==
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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.
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===Reagents===
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Biological resources e.g. cell lines, buffers (link to a method for making them), enzymes, chemicals, kits, etc.
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===Equipment===
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Any equipment used to perform the protocol (link to a method for using them).
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==Procedure==
==Procedure==
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A step by step guide to the experimental procedure.
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*First you need to purify the DNA you want to analyse (see for example [[Chromosomal DNA isolation from E. coli]]).
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*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).
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*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.
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*add 90μL dH<sub>2</sub>O
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*use 10μL as template for 1 PCR reaction
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*for the qPCR use primers ter (for terminus) and 3921366 (for oriC). Details are here: [[User:Torsten_Waldminghaus/Primers]].
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*Make primer mixes for each set of primers:
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If you find it helpfull you could use the following icons to highlight important parts:
 
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[[Image:Difficult step.png]] icon to highlight difficult steps
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{|border="1"
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|+ '''Primer Mix'''
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!for 250μL
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|-
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|22.5μL primer fw (100pmol/μL)
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|-
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|22.5μL primer rv (100pmol/μL)
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|-
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|6.25μL probe (100pmol/μL)
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|-
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|198.75μL ddH<sub>2</sub>O
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|}
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[[Image:Critical step.png]] warn about critical steps
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*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)
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#12.5μL TaqMan Mix
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[[Image:Time required.png]] help people plan how long steps will take
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#2.5μL primer mix (see table above)
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*pipett 15μL in wells on 96-well PCR plate
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[[Image:Pause point.png]] where protocol can be interrupted
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*add 10μL DNA
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*spin down plate at 1000 rpm to collect sample on the bottom
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[[Image:Optional step.png]] steps that can be omitted or included
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*run qPCR
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*calculate oriC/ter ratio normalized to the oriC/ter ratio of rif sample
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==Critical steps==
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Referenced from the main protocol, a more thorough explanation of particularly important steps in the protocol.
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==Notes==
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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>
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Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.<br>
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==Discussion==
==Discussion==
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You can [[Talk:{{PAGENAME}}|discuss this protocol]].
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You can [[Talk:{{PAGENAME}}|discuss this protocol]].
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Tag this page with categories to allow easier indexing and searching.  See [[Categories]] for information on existing categories.
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Current revision

Contents

Curators

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 discussing the idea of protocol curators. Please contribute.

Abstract

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

  • 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. 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: 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 dH2O
  • 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.
  • Make primer mixes for each set of primers:


Primer Mix
for 250μL
22.5μL primer fw (100pmol/μL)
22.5μL primer rv (100pmol/μL)
6.25μL probe (100pmol/μL)
198.75μL ddH2O
  • 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)
  1. 12.5μL TaqMan Mix
  2. 2.5μL primer mix (see table above)
  • pipett 15μL in wells on 96-well PCR plate
  • add 10μL DNA
  • spin down plate at 1000 rpm to collect sample on the bottom
  • run qPCR
  • calculate oriC/ter ratio normalized to the oriC/ter ratio of rif sample


Discussion

You can discuss this protocol.

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