Sizing of E. coli plasmids- HELP!!

ja.child at ukonline.co.uk ja.child at ukonline.co.uk
Thu Oct 15 08:24:23 EST 1998


I'm getting in a bit of a mess over sizing of plasmids in wild-type isolates of E.coli.
Can someone wiser out there help me please?

Am I right in thinking that:
1. a linear DNA ladder can ONLY be used to size linear (e.g. digested) DNA fragments?

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.

If this is the correct way of doing it, doesn't shearing of plasmids affect the result?  (How can you tell from a plasmid gel whether this has happened?)

Can you accurately size plasmids this way, or should they ideally be linearised first?  

Thanks,

Jenny



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