OriC/ter ratio determination
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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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- 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.
|22.5μL primer fw|
|22.5μL primer rv|
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