Summary of Plasmid Rescue Responses

Department of Molecular Karma YABLONSKY at BIOVAX.RUTGERS.EDU
Thu Jul 5 09:17:00 EST 1990


Hello everyone!!!

I'm sorry It has taken so long for me to get this summary together but the
bench calls...

Below is my original post and the responses I recieved.  I had a student try
a double exonuclease procedure (lambda exo followed by exo VII).  This did
reduce the amount of total DNA is the sample but we did not see any increase
in the amount of rescued plasmids.  We did however see less competition of the
sample with the added control plasmids.  This effect was not due to the exo
treatments alone. Some relief was gained just by running an undigested sample
through an additional phenol/chloroform extraction and a second spun column.

-----------------------------


From:	MBCL::YABLONSKY    "Department of Molecular Karma" 24-MAY-1990 12:33:50.46
To:	IN%"METHODS-AND-REAGENTS at genbank.bio.net", IN%"biosci at genbank.bio.net", IN%"bionet at genbank.bio.net"
CC:	YABLONSKY
Subj:	plasmid rescue experiments


I'm experiencing a problem with a plasmid rescue experiment. It appears
that the overwhelming amount of host cell DNA isolated in the total DNA
preparation effectively masks most of the rescuable plasmids when 
transforming E. coli DH10B (BRL).  I am sure that this is occurring 
based on experiments in which I dose the total DNA preparation with some
control plasmid before transformation.  These control plasmids are also
masked. The effect seems to be due to the fact that relatively
high amounts (10s of ng ) of total DNA must be used in transformation 
in order to see any rescue at all.  

I have tried further purification of the total DNA preparation
via spun-columns and see no relief of this effect. I am considering
trying to eliminate the linear DNA from the total DNA preparation
via exonuclease or alkaline treatment. I thought that I would post
this query first hoping for some good advice before I waste some of
these DNA preparations. If anyone has any experience in plasmid rescue
or some other enlightening comments please e-mail me directly. 

Thank you,

 Michael D. Yablonsky                  internet: yablonsky at biovax.rutgers.edu


----------------------------

From: "Silverman, Sanford" <silverman at fcrfv1.ncifcrf.gov>
Subject: RE: plasmid rescue experiments
To: yablonsky <yablonsky at biovax.rutgers.edu>

I've had similar problems rescuing plasmids from yeast dna.  You could try
using different efficiency competent cells or a different commercial source
of same.  I would also try some nondestructive technique such as CsCl 
gradients to separate your plasmid before resorting to nucleases, etc.
Hope my $0.02 was helpful; good luck.------Sandy

------------------------------

From: patrick linder <linder at urz.unibas.ch>
Subject: plasmid rescue experiments
To: Department of Molecular Karma <YABLONSKY at biovax.rutgers.edu>
In-Reply-To: <9005241638.AA01647 at genbank.bio.net>
Message-Id: <1177:linder at urz.unibas.ch>

We experience different transformation rates with yeast minipreps depending
on the E.coli strain we use. May be you shoud use other strains and/or other
transformation procedures, i.e electroporation.
Let me know if you find out, how to do it.
Good luck,
Patrick Linder


---------------------------------

From: chiafari at umbc3.umbc.edu
Subject: Re: plasmid rescue experiments
To: YABLONSKY at biovax.rutgers.edu
Message-Id: <9005251259.AA28011 at umbc3.umbc.edu>
Newsgroups: bionet.molbio.methds-reagnts
In-Reply-To: <9005241638.AA01647 at genbank.bio.net>
Organization: University of Maryland, Baltimore County

Have you run transformation cotrols to determine the transformation efficency?
Maybe you have a bad lot of cells. I haven't done any plasmid rescue in about
2 years, but i don't remember anything like your describing.Is the plasmid you
are rescuing linear and integrated in the mamalian genome (where you would have
to rely on recombination), or is the plasmid capable of replication in Eukary-
otes (because of viral sequences). The former is much more difficult than the 
latter. Your going to need alot of starting material if you want to purify by
column, because of non-specific binding. Is your prep contaminated with an 
endonuclease? is there lots of high molecular weight DNA in it?

Just some things you should think about.

-frank

-- 
Science and engineering must be linked, or science degrades into philosophy...
	Francis A. Chiafari  -Molecular Biologist- Balt. Rh Typing Lab
			        w (301)225-9595      h (301)461-4874

---------------------------------

From: SHEPHERD%ESVAX at dupont.com
Subject: RE: plasmid rescue experiments
To: YABLONSKY at biovax.rutgers.edu
X-VMS-To: ESPRNT::IN%"YABLONSKY at biovax.rutgers.EDU"

When you get the answer(s) please forward.  Thanks.

-----------------------------

Date: Tue, 29 May 90 13:27:36 EDT
From: agostino at med.unc.edu
Subject: plasmid rescue
To: yablonsky at biovax.rutgers.edu
Message-Id: <9005291727.AA16654 at maiden.med.unc.edu>

Hi,
	I struggled with plasmid rescue a while back and determined that my
main problem was not the contaminating genomic DNA but the contaminating
SDS used to lyse the cells.  I reduced the SDS and my Tx efficiency went up
but still not very high.  The fellow I work with suggested an additional
extraction that removes SDS--that is, a 1:1 mixture of chloroform and
ethyl acetate.  This increased the Tx efficiency at least 10 fold.
mike agostino
unc chapel hill

-------------------------------------


From: agostino at med.unc.edu
Subject: RE: plasmid rescue
To: YABLONSKY at biovax.rutgers.edu
Message-Id: <9005301533.AA16933 at maiden.med.unc.edu>

Glad you think the extraction is a good idea.  I EtOH precipitated as usual
with a 70% EtOH wash of the DNA pellet.  Ethyl acetate is an organic liquid
so I used it "straight" with chloroform at a 1:1 ratio (kept it in a dark
bottle, 4C, under TE, just as you would phenol.  Please tell me if it 
works for you (and if it doesn't I'll try again!)
mike


-------------------------

Mike, I haven't tried this as yet. I thought to add this as an extra cleanup 
after the exo treatments. It didn't get done because the student was running 
out of time. As of now the student is out of the lab for the rest of the 
summer.  I'll have them do this again when they return.


Again, thanks to everyone...


 Michael D. Yablonsky                  internet: yablonsky at biovax.rutgers.edu
 Department of Molecular Karma          at&tnet: (201) 932-3052
 Waksman Institute
 Rutgers University                    A good planet is hard to find!
 Piscataway, NJ, 08854-0759




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