Path: utzoo!utgpu!watserv1!watmath!uunet!samsung!noose.ecn.purdue.edu!iuvax!purdue!haven!uvaarpa!murdoch!krebs.acc.Virginia.EDU!mn5y
From: mn5y@krebs.acc.Virginia.EDU (Mukund Nori)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Ligations
Message-ID: <1990Sep10.172514.26901@murdoch.acc.Virginia.EDU>
Date: 10 Sep 90 17:25:14 GMT
References: <9009082352.AA08801@genbank.bio.net>
Sender: news@murdoch.acc.Virginia.EDU
Organization: University of Virginia
Lines: 26

re: Q1:  Yes, the plasmid will grow in the bugs = DH5 alpha from BRL; am
using the Hanahan protocol for transforming them.

re: Q2:  Yes, I can transform the uncut plasmid into the bugs; that's how I
made a big plasmid prep.  I am using amp plates for selection.

re: Q3:  I am using approx 100 ng in 20 ul total reaction volume.  Conditions
range from 37 degrees for 4 hours to 15 degrees for 18 hours; using 10 ul
[=0.5 of reaction] to transform 100 ul of bugs and spreading 1/10 onto
amp plates.  The plasmids are being cut initially with either BamHI or ClaI.
The DNA is purified from agarose using the freeze-squeeze method and cleaned
using Tris-sat phenol; phenol-chloroform-isoamyl alcohol; chloroform-isoamyl
alcohol; ethanol ppted and resuspended in 30 ul TE.

I understand that these are not quite as trivial as they sound.  The same 
conditions work like a charm for people across the hall.  I am doing a test 
using my DNA with all their reagents.  Hope to know the answer tomorrow.

I don't know that there is a difference between speculation and educated [:-)]
guesses.  Thanks in advance for any suggestions.


 ******************************************************************
 ___Raistlin___  			Mukund Nori
 Raistlin@Virginia.EDU			mn5y@krebs.acc.Virginia.EDU
      "VIOLENCE IS THE LAST RESORT OF THE INCOMPETENT" Asimov