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Tips on how to plasmid rescue p{PZ}?

Robert M. Mihalek mihalek at FORMULA1.smtp.anes.upmc.edu
Thu Jun 24 09:57:36 EST 1999

In article <19990624002958.17825.qmail at hotmail.com>
ze_cheng at HOTMAIL.COM (ze cheng) writes:

> Hi,
>     I am a graduate student of SUNYAB.My research project involves plasmid 
> rescueing p{PZ} from p-element containing stains.I tried several times in 
> vain so far,even with the combination of digestion with XbaI and NheI or 
> XbaI with SpeI.The problem here is that I have to clone a very big 
> plasmid(at least 7Kb).Has anyone succeeded in doing that?Can someone give me 
> some tips on how to ligate and transform effiently?Thank you very much.

I'm sure you have a good restriction map of the P-element, but since
you're having problems, you may want to double-check the restriction
map to make sure that your choice of enzymes will actually remove the
plasmid sequences in the P-element along with the flanking genomic DNA.

Once you have (re) verified that the enzymes are correct, the only
trick I can think of is to dilute your ligation reactions. Since you
want to select preferentially for an intramolecular ligation (i.e.
circularization of the rescued plasmid), diluting the ligation reaction
will make intermolecular ligations less likely.

7kb is not a very big plasmid, and is not the cause of the problem
(when you get to around 15-20 kb, you'll have some problems).

Bob Mihalek=================================================
please remove the name of the RACE SERIES for e-mail replies
"I have neither been there nor done that." Bart Simpson

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