HELP : Blunt to Sticky ?

Bernard Murray bernard at elsie.nci.nih.gov
Mon Jan 23 00:05:18 EST 1995


In article <1995Jan19.144238.7472 at nki.nl>, gbrink at nki.nl writes:
> Dear All,
> 
> Who can tell us how we can link one T to a blunt-end DNA fragment to create
> a sticky T-overhang.
> 
> Guido Brink


I assume that you are making your own T vector (or something similar).
James George posted the a "how to do it" and I include the relevant
section below.  He started with 10 ug pUC19 and cut it with SmaI.
	I hope that this is what you want.
		Bernard

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov  (National Cancer Institute, NIH, Bethesda MD, USA)

[Included section starts below]
------------------------------------------------------------
This procedure is adapted from D. Marchuk, M. Drumm, A. 
Saulino, and F.S. Collins Nuc. Acids. Res. (1991) 19:1154. 

*********************T-TAILING THE VECTOR****************** 
 
At this point, it is assumed that there has been 80% 
recovery of the cut plasmid DNA. 
 
1. Resuspend the plasmid DNA in 63ul water (conc approx. 130ng/ul) 
2. To the resuspended plasmid add: 
 
10ul 10X PCR buffer (standard cetus stuff, no MgCl) 
20ul 10mM dTTP [2mM final] 
 6ul 25mM MgCl2 [1.5mM final] 
 1ul Taq polymerase (Cetus amplitaq 5U/ul) 
______ 
100ul total volume. 
 
3. Incubate for 3 hours at 70 degrees C. 
4. Extract with Phenol, Phenol/chloroform, chloroform. 
5. Extract twice with ether (so I'm paranoid!) 
6 add 75ul 2M **ammonium** acetate (assuming 75ul recovery from extractions). 
7 Add 150ul isopropanol. Spin 20mins in microfuge at full speed at 4 degrees. 
8. Wash with 70% ETOH 
9 Dry pellet in spin vac and store at -20 degrees until use.


Original bionet post by
==============================================================================
James F. George, Ph.D.              "Back off man, I'm a scientist"
Department of Surgery                --Bill Murray
University of Alabama at Birmingham
205-934-4261 voice
txpljfg at uabcvsr.cvsr.uab.edu
===============================================================================




More information about the Bioforum mailing list