colony PCR for E.coli genomic sequences

R.D. Haigh rxh at leicester.ac.uk
Tue Dec 9 14:03:01 EST 1997


Hi Richard
I realise that this may be a little too late in terms of your 
constructions etc. but we certainly have found the best way to make 
mutants which will have no phenotype is to use a positive selection 
system (as you appear to be in E. coli the most obvious is SacB). 
If carefully handled this can give you upto 50% mutants which is 
hardly too stressful in terms of screening by colony PCR. If you are 
interested I have several vectors which will work in coli (depending 
on how wild it is) and one may be suitable for the mutant you 
have (or intend to make) - positive selection is much faster than any 
screening method I've seen so far.

Richard


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    Richard Haigh                      
    Department of Microbiology and Immunology,                        
    University of Leicester,
    University Road,        
    Leicester. LE1 9HN UK
  
    Phone: 0116 2523017 
    FAX   : 0116 2525030 
    Email : rxh at nospam.le.ac.uk
    NB. remove nospam to Email  
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