Plasmid isolation

Mikhail Alexeyev malexeyev at biost1.thi.tmc.edu
Thu Feb 29 12:28:20 EST 1996


In article <3134CC22.3C43 at biology.watstar.uwaterloo.ca>,
syeh at BIOLOGY.WATSTAR.UWATERLOO.CA wrote:

> I have a problem isolating plasmid from a recombinant bacteria 
> expressing a foreign gene. Can you suggest me ? The bacteria is 
> DH5-alpha, plasmid is pUC19, cloned gene is a gene from Enterobacter. 
> The phenotype of the recombinant bacteria changed, for example the color 
> became purple after having this foreign gene. I tried alkaline lysis, 
> followed by phenol:chloroform extraction and precipitation either by 
> alcohol or by isopropanol and then PEG. No plasmid obtained. I tried 
> also Promega's Wizzard rasin. No plasmid again. 
>   Please suggest me. My email: s3shah at sciborg.uwaterloo.ca
> Thanks in advance. saleh Shah


You may try to shorten the procedure (use only solutions I, II, III and
EtOH or isoprop. precipitation). That would eliminate possibility that DNA
is lost during PCI treatment or PEG precipitation. If you wouldn't see
anything on the gel even then, you may consider the possibility that you
you are dealing with some sort of contamination (purple colour is not
characteristic of E. coli, unless your insert codes for some colored
product). You may try to restreak you bacteria on other antibiotics
(streptomycin, 20 ug/ml; kanamycin, 30 ug/ml; tetracyclin, 15 ug/ml,
etc.). If you bacteria will appear to be resistant, then you are most
probably dealing with contamination. 

I personally would repeat transformation.

M. Alexeyev



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