P1

Bruce Roe broe at aardvark.ucs.uoknor.edu
Fri Aug 27 07:04:00 EST 1993


In article <1993Aug23.204227.7560 at news.cs.brandeis.edu>, cbrandes at binah.cc.brandeis.edu writes...
> Does anybody have a protocol for P1 vectors?
>I would prefer it if it actually gave me a yield!
> 
>Thanks in advance
>				Scotty
Hi,
        Any standard isolation technique that one usually uses for
plasmid or cosmid preps should work.  For example:
A cleared lysate followed by PEG pptn and CsCl centrifugation.

The problems with P1 that we have found are:
        1. don't forget to induce with IPTG after the cells reach
late log.
        2. remember you are dealing with a large DNA so be
very careful not to physically shear it (need to be much more
gentle during the isolation than you would for plasmids or cosmids.
        3. the yield usually is lower than with smaller cosmids and
plasmids (sometimes 10x lower) and seems to vary depending on what
has been cloned into the P1 vector.
	4. be sure to begin with cells from a fresh plate, i.e.
beginning with petri dishes with cells containing P1's that have been
stored at 4deg for more than a week seems to give lower cell growth
in liquid media and thus lower yield (probably a longer lag phase).
	5. check that you are using the correct antibiotic at the
recommended concentration for petri dishes and liq. media.

Good luck and take care........bruce
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