Fill-in enzymes

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Tue Jul 27 02:13:00 EST 1993


Hi Greg!
I have always used DNA pol I Klenow fragment to fill 5' overhangs. 
I have used it to blunt end
clone many different restriction fragments and in each case (can 't remember
a time when it didn't work properly) it regenerated the restriction sites
that should have.  When i didn't check before i cloned occasionally 
I was suprized to find one or both of the terminal restriction sites 
regenerated
just as they should have been.  Did you know that if you
blunt end clone a ClaI fragment into an EcoRV site you regenerate your ClaI 
sites
at both ends.  I have had a number of suprizes in my years of blunt-ending
with Klenow but all have been from my over sight and not from nucleotides 
removed.
I also use klenow to blunt gene fragments into frame for the production of 
fusion 
proteins.  So far no complaints! 
The reaction conditions are 
        200ngs of vector DNA
        1x nick translation buffer made with MgCl2 not Mgsulfate (for ligase)
        100 uM dNTPs
        approx 3 molar excess of restriction fragment
        1-5 units of Klenow
       volume to 20ul
    Incubate at rt for 15 -20 min
For a ligation a just add
        0.5ul of 250mM DTT
        0.5ul of 50mM ATP   
        1ul of T4 DNA ligase (5 units/ul from Beohringer Mannheim)
Incubate 12 hrs at 10-12oC.

Good Luck

Dan Gietz
______________________________________
R.Daniel Gietz Ph.D.
Assistant Professor
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm 250
Winnipeg, Manitoba, Canada
R3E 0W3
Tel.: (204)789-3458
Fax.: (204)786-8712
E-mail GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
"Trying to do the Manitoba Thing"
_______________________________________


------------------------------------------------------------------------------
REPLY FROM: Gietz, Dr. Dan
Return-Path: <BIOSCI-REQUEST at net.bio.net>
Date: Mon, 26 Jul 93 11:04:49 PDT
From: greg at mendel.llnl.gov
Message-Id: <9307261804.AA00269 at mendel.llnl.gov.>
Reply-To: Greg Lennon <greg at mendel.llnl.gov>
To: methods at net.bio.net
Subject: Fill-in enzymes
------------------------------------------------------------------------------

We are searching for an enzyme that can reliably fill-in
5' overhangs such that they become blunt ends ... but
without ever losing even a base off the end.  Our luck so
far, even with 3'-5'exo lacking enzymes, has not been
great.  Would anyone have recommendations concerning either
the enzyme of choice and/or optimal reaction conditions ?

Many thanks,

Greg Lennon

****************************************************************

 
Greg Lennon
Human Genome Center, L-452
Lawrence Livermore National Lab
Livermore, CA 94550   USA
Email: greg at mendel.llnl.gov
Phone: (510) 422-5711  Fax: (510) 423-3608



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