YABLONSKY@BIOVAX.RUTGERS.EDU (Department of Molecular Karma) (07/05/90)
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@genbank.bio.net", IN%"biosci@genbank.bio.net", IN%"bionet@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@biovax.rutgers.edu ---------------------------- From: "Silverman, Sanford" <silverman@fcrfv1.ncifcrf.gov> Subject: RE: plasmid rescue experiments To: yablonsky <yablonsky@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@urz.unibas.ch> Subject: plasmid rescue experiments To: Department of Molecular Karma <YABLONSKY@biovax.rutgers.edu> In-Reply-To: <9005241638.AA01647@genbank.bio.net> Message-Id: <1177:linder@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@umbc3.umbc.edu Subject: Re: plasmid rescue experiments To: YABLONSKY@biovax.rutgers.edu Message-Id: <9005251259.AA28011@umbc3.umbc.edu> Newsgroups: bionet.molbio.methds-reagnts In-Reply-To: <9005241638.AA01647@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@dupont.com Subject: RE: plasmid rescue experiments To: YABLONSKY@biovax.rutgers.edu X-VMS-To: ESPRNT::IN%"YABLONSKY@biovax.rutgers.EDU" When you get the answer(s) please forward. Thanks. ----------------------------- Date: Tue, 29 May 90 13:27:36 EDT From: agostino@med.unc.edu Subject: plasmid rescue To: yablonsky@biovax.rutgers.edu Message-Id: <9005291727.AA16654@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@med.unc.edu Subject: RE: plasmid rescue To: YABLONSKY@biovax.rutgers.edu Message-Id: <9005301533.AA16933@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@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