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]]. 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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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]]).
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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. 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).
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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).
*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
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!for 250μL
!for 250μL
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|-
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|22.5μL primer fw
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|22.5μL primer fw (100pmol/μL)
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|-
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|22.5μL primer rv
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|22.5μL primer rv (100pmol/μL)
|-
|-
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|6.25μL probe
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|6.25μL probe (100pmol/μL)
|-
|-
|198.75μL ddH<sub>2</sub>O
|198.75μL ddH<sub>2</sub>O

Current revision

Contents

Curators

Torsten Waldminghaus

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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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