Re consistent lambda preps

[Antje Haase]

From: antje (Antje Haase)
Message-Id: <9307260437.AA16143 at rt1.menzies.su.edu.au>
Subject: Re: Consistent Lambda preps
To: hdang at cns.neusc.bcm.tmc.edu
Date: Mon, 26 Jul 93 13:37:37 CST
Cc: methods at net.bio.net
In-Reply-To: <9307260301.AA18198 at net.bio.net>; from "Hong Dang" at Jul 26, 93 2:55 am
X-Mailer: ELM [version 2.3 PL11]
 
 
 
> > Dear Netters:
> > 
> > I would very much appreciate some pointers on CONSISTENT Lambda preps. My wife
> > is having a hard time getting consistent yield of a lambda clone containing a 
> > about 18 kb insert. Is there a FAQ on this subject? If yes, where can I find it?
> > Any help would be very much appreciated. Thank you in advance!
> > 
> > Hong
> > 
> > hdang at channel.neusc.bcm.tmc.edu
> > 
> > 
Hi Hong,
 
 when I was doing  a lot of lambda preps I found that it is most
 important to have a very well lysed culture in the first place. It's got to
 be almost transparent with protein flakes floating everywhere. If you don't
 get that it is not worth starting a DNA prep at all. You may set up several
 cultures in parallel using various amounts of cells with a constant amount of
 phage. Phage taken from fresh plaques work best! 
 The other important thing is aeration, make sure you have a big anough flask
 for the culture you are growing. Often the yield is higher from a smaller
 culture: 50ml culture (in 500 ml flask) gives you up to 1mg of lambda DNA. 
 
 DNA prep itself is straightforward, basically following the "standard method
 for purification of Bacteriophage lambda" in "Maniatis" up to the PEG
 precipitation and centrifugation. It needs about 50
 ug DNAse and RNAse for the initial step though (not 1ug). Don't do the CsCl
 gradient but simply resuspend the pellet in SM/20mM EDTA/0.5% SDS, add
 50ug/ml proteinase K, incubate 1hr 65C, phenolize, EtOH precipitate. For best
 quality DNA don't spin but "fish out" your DNA with a pipette tip.
 Always works very well and gives high quality DNA! 
 
 Good luck
 
 Antje  
 
 
 
 ______________________________________________________________________________
 Antje Haase
 Menzies School of Health Research
 Casuarina, NT 0811
 Australia
 _______________________________________________________________________________