HELP : Blunt to Sticky ?
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 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
txpljfg at uabcvsr.cvsr.uab.edu
More information about the Bioforum