cosmid prep info wanted

Bruce Roe broe at aardvark.ucs.uoknor.edu
Wed Sep 22 07:28:00 EST 1993


In article <1993Sep21.112558.7905 at wvnvms.wvnet.edu>, lsalati at wvnvms.wvnet.edu writes...
>Hi,
> 
>I received a cosmid clone from another researcher and would like some
>suggestions on methods used for amplification and preparation of more
>DNA.  I have never worked with cosmids before so I thought I'd ask here.
> 
>The cosmid is pCOS2EMBL with an insert of about 50 kb.
> 
>1) Is there a preferred bacterial strain for growing cosmids or are
>   they dependent on the vector involved?  We currently have DH5-alpha,
>   JM109 and JM101 and SURE cells in the lab.

Use extreme caution here. Cosmid clones tend to rearrange and/or lose
some or all of the insert.  Contact the folks who did the original cloning
and get their strain, and BE SURE it is rec minus.  I don't have handy
our list of strains, but DO NOT use those you've listed above.  You may
be lucky but don't take the chance of strange things happening to your
insert.

> 
>2) What method is used to transform bacteria with cosmid DNA?
>   Electroporation?

Standard calcium treated competent cells work, but not very efficiently.
Electroportation also works with higher efficiency.  Since you are just
trying to transform with a single cosmid, not making a library, we use
standard calcium treated cells.

> 
>3) How careful do you have to be when preparing DNA from the bacteria?
>   Since the DNA is large I expect that I have to more careful to prevent
>   breakage.  Are there any changes from a standard plasmid prep other
>   than being careful in handling the lysate?

More careful than when isolating chimeric plasmids.  40-50 kb is pretty
large (when compared to 5-10 kb plasmids) so don't vortex, just gently
mix by hand, and try to eliminate steps that would produce any shearing.

> 
>Any help is much appreciated.

Although you didn't ask, the yields of cosmids per liter of cells, seems
to vary depending on the host, nature of the insert, and the vector itself.
Don't be supprised or depressed if you only get 1-200 micrograms or less
cosmid per liter.  I'd also suggest doing a growth curve and not letting
the cosmid containing cells grow into late log phase before harvesting.
The longer some of these cells grow, the more the tendency for strange
rearrangements and deletions to occur.  You may be lucky but then again
maybe not, so keep the growths at 6-8hours (not over night) and don't
do more than a 1:50 dilution for transfers from small volume to large
volume media.  We do, colony to 3 ml TB+antibiotic for 6 hours, then
the 3ml into 50 ml of TB+antibiotic for another 6 hours and finally,
50 ml to 1 liter TB+antibiotic for another 6 hours.  Try to err on the
side of caution, we do only because we've been burnt several times.

> 
>Stephen Klautky
> 

Hope this helps.......bruce
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