Reasons for poor plasmid showing

j.r.stephen mbi071 at sysc.abdn.ac.uk
Tue Dec 17 07:07:54 EST 1996


Hi Ed,

Just incase you don't get any further with this, you can always try a PCR on
your clones using the SP6/ T7 priming sites (just pick a tiny amount of colony
and add it to your reaction). You should get a band of the size of your expected
insert plus about 120 bases of cloning site. If you do, then there must be
something wrong with your prep. Possibly you are leaving the lysate in the lysis
solution too long, or you are using endA+ E.coli hosts. If the latter, I believe
Promega have a new prep specifically designed for retrieving plasmids from endA+
hosts which contains an alkaline protease to destroy the offending enzyme. No
affiliations etc....

Have fun!

John

ed marsden (ed.marsden at utoronto.ca) wrote:
: 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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