Sma I religation problem
chrisb at festival.ed.ac.uk
Tue Jul 25 04:07:27 EST 1995
Amadou Ba (PHYASB) wrote:
: Adding Klenow and dNTP would make the overhang in the other end
: blunt and the insertion may occur in both direction ?
: We're doing EcorI and SmaI to insert a fragment digested with
: EcorI and StuI (blunt end).... Each enzyme linearize the corresponding
: vector when used alone. But like in the original article we get to
: many colonies from the control ligation (no insert).
: If we understand correctly, adding Klenow and dNTP will make
: every thing blunt ended.
The trick is to change the order of events in preparing the vector:
(1) Cut with SmaI / heat kill enzyme
(2) Polish the ends with T4 DNA polymerase (NOT Klenow*) and excess dNTPs /
heat kill enzyme
(3) Cut with EcoRI / heat kill enzyme
(4) Clean up DNA (best to gel purify to remove uncut material and polylinker).
* TI: POLISHING WITH T4 OR PFU POLYMERASE INCREASES THE EFFICIENCY OF
CLONING OF PCR FRAGMENTS
AU: COSTA_GL, WEINER_MP
JN: NUCLEIC ACIDS RESEARCH, 1994, Vol.22, No.12, p.2423
Then your vector will be as desired.
Chris Boyd | from, \MRC Human Genetics Unit / Western General Hospital
chrisb at hgu.mrc.ac.uk| not for \ Crewe Road / Edinburgh EH4 2XU / Scotland
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