molecular typing of transconjugants

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Alexandra Moura

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Oct 12, 2008, 8:40:29 PM10/12/08
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Dear all,

 

I’m planning to obtain a huge set of E.coli transconjugants and I was wondering if there is

any way to perform molecular typing of E.coli transconjugants besides restriction analysis of DNAp?

     

 

The gold would be to reduce the amount of isolates (less expensive and time consuming…) for further plasmid characterization.

 

 

Would typing tecniques such as rep-PCR with crude cell extracts work to exclude identical transconjugants?

 

 

Any suggestions would be very welcome!

 

Kind regards,

 

Alexandra

 

Christopher Thomas

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Oct 13, 2008, 3:01:26 AM10/13/08
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Dear Alexandra

I guess it depends on what you are trying to achieve.  I assume that you are selecting for certain markers being brought into E. coli by conjugation.  I think that the first thing would be to isolate plasmid DNA from a manageable number of colonies (eg 24 or 48) and see how diverse they are – if they are all different then this would lead to one strategy while if there are many the same then it would lead to a different strategy.  It also depends on whether you want to isolate plasmids of as many different Inc groups as possible or whether you want to take a representative distribution of the plasmids that are there.  If you are trying to isolate plasmids of a specific group then screening pools by PCR would save you time.  If you are looking for plasmids that are different from the dominant type then you could do the same but screen the DNA by electrophoresis and look for pools that have extra weak bands.  Will be happy to discuss more if necessary.

Best wishes

Chris Thomas

 

Christopher M Thomas

Director of the University Graduate School

Professor of Molecular Genetics

School of Biosciences

University of Birmingham

Edgbaston

Birmingham

B15 2TT

 

Phone +44 121 414 5903

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Alexandra Moura

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Oct 13, 2008, 5:50:28 PM10/13/08
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Dear Chris,

 

Thank you very much for your suggestions. I think I’ll start by typing a ‘manageable number of colonies’ and see how it works.

 

Thanks for your help!

 

Kind regards,

 

Alexandra

 

 

 

 

 

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