OriC/ter ratio determination

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*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
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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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Revision as of 04:55, 27 February 2009

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 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

  • 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: 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.
Primer Mix
for 250μL
22.5μL primer fw
22.5μL primer rv
6.25μL probe
198.75μL ddH2O


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

Image:Difficult step.png icon to highlight difficult steps

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Image:Pause point.png where protocol can be interrupted

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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 *'''~~~~''': in front) and explained. Links to FAQs/tips provided by other sources such as the manufacturer or other websites would be best made here.
Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.


Discussion

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