Sizing of E. coli plasmids- HELP!!

Rafael Maldonado rafamaldo at geocities.com
Mon Oct 19 05:19:55 EST 1998



ja.child at ukonline.co.uk wrote:

> 1. a linear DNA ladder can ONLY be used to size linear (e.g. digested) DNA fragments?
>

Yesm you are correct.


>
> 2. Undigested plasmids in E. coli can be sized by comparing the size with those from a standard marker strain such as the NCTC 50192 and 50193 in the UK.  You would do this by plotting a calibration graph of  log (plasmid MW in Md) against distance migrated from the well, and read off the MW of the plasmids you want to size from this graph.

I never sized not linear DNA fragments, and I don't know the markers you refer, BUT a plasmid will almost never show only one form, depending of the topology of the molecule: supercoiling, concatemerization, oligomerization... Also, during the extraction, the DNA change the conformation, adding complexity to the pattern in a gel. I guess you
can use those markers for circular plasmids with no supercoiling (nicked), but your problem will be to know which form is the circular one among the several bands you see from only a plasmid. Difficult task.

My advice: linearize the palsmids and use a regular linear DNA marker.

-Rafa


--
Rafael Maldonado
Division of Genetics
University of Alicante, Spain
rmaldonado at ua.es





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