Reasons for poor plasmid showing

David Law dave at gargoyles.demon.co.uk
Sat Dec 14 20:55:24 EST 1996


	Your plasmid should contain a selectable marker, ussually antibiotic
resistance genes ( i.e ampicillin resistance). Streak a colony of your
clones on plates containing the apropriate antibiotic : growth indicates
the presence of the plasmid in your clones.

If your doing the plasmid extraction correctly, there should be very little
RNA or chromasomal DNA in your mini-prep.

	BASIC STEPS.
1 Harvest approx. 5mm cubed of bacterial cells (cleaned).
2 Add NaOH / SDS 
3 Add KCl 
4 Incubate at ~4 degree C for 5 min.
5 Centrifuge and collect supernatant.
6 Add phenol/chloroform to supernatant (mix well).
7 Centrifuge and collect supernatant.
8 Add ethanol to supernatant.
9 Centrifuge and discard supernatant.
10 Dry remaining pellet and resuspend in small volume of T.E contaning
RNase.

Look in Sambrook et. al (cloning manual) for exact protocol.

pUC cloning vectors contain replication origons that result in a high copy
number.

Plasmids do not generally hybridize strongly with the chromasomal DNA (due
to there super coiled nature) and even if they did a high copy number
plasmid will probably have over 100 copies per cell the majority will
certanly not hybridize.
 
I've recently finished a third year molecular genetics research project
where my biggest problem was transformation frequencies so good luck
(you'll probably need it).

-- 
David Law

Patient-- Docter, Docter people keep ignoring me.

Docter-- Next please.

P.S Whats a "watewater".

ed marsden <ed.marsden at utoronto.ca> wrote in article
<32B0F0B2.771C at utoronto.ca>...
> 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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