extremely low copy number plasmid

Dr N.I. Leaves nleaves at hgmp.mrc.ac.uk
Thu Jan 31 05:10:08 EST 2002


On Wed, 30 Jan 2002, Alison Leakey wrote:

> Hello everyone!
>
> I would appreciate any suggestions. I am working with a plasmid that is
> in extremely low copy number. Lately we have been trying to increase the

<snip>

Hi Alison,

are you sure you've actually got extrachromasomal plasmid. There
are naturally occuring plasmids that chromsomally integrate and therefore
would not appear after an alkaline lysis prep. I used to work with
Haemophilus which could be multiresistant to antibiotics. Filter matings
into a rec- recipient can trap these plasmids extrachromasomally and make
them visible in alkaline lysis extractions - otherwise no show. Once we
trapped the plasmid we developed a probe and PCRs to detect the
chromasomally integrated forms (we published this stuff in J Antimicrob
Chemother in 2000)

If your plasmid is truely free and in low copy then what do you want to do
with the DNA when you've got it? If you just wanted to sequence or to
make PCR template you could try a new product I've come  across from
Amersham (no affiliation) called templiphi. Its a rolling circle
amplification which is inoculated directly with culture.

Hope some of this helps, good luck

nick

end
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Dr N I Leaves
Mouse Sequencing
MRC HGMP Resource Centre
Hinxton
Cambridge CB10 1SB
tel: 01223 494557 (office) or 01223 494541 (lab)
email: nleaves at hgmp.mrc.ac.uk
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