Reasons for poor plasmid showing

ed marsden ed.marsden at utoronto.ca
Fri Dec 13 00:59:14 EST 1996

Hi everyone. I've been attempting to isolate recombinant pUC 19 from several different 
E.coli clones using the NaOH / SDS method. The clones were provided from another student 
who had created a library last year from a watewater strain. Here's the problem:

The first time I had run agarose gels, there were no plasmids. Subsequent experiments 
had demonstrated that my method could still free up RNA and globs of DNA. I had run a 
standard pUC in one lane for the purpose of comparing molecular weights but no other 
isolates had shown bands in that region. I have come up with only four answers:

(1) The clones do not harbour any plasmids 
(2) The copy number of the existing plasmids is too low to quantify on a gel
(3) The plasmids in question are hybridizing with chromosomal DNA
(4) I am an incompetant with regards to plasmid isolation

	Any suggestions would certainly be appreciated. I'm about to consider 
transforming a strain myself to ensure that the vector is in the cells instead of going 
on a student's word.

							Yours truly,  Neil M.

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