Sma I religation problem

[Chris Boyd]

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.

Best wishes,

Chris Boyd          | from,  \MRC Human Genetics Unit / Western General Hospital
chrisb at hgu.mrc.ac.uk| not for \        Crewe Road / Edinburgh EH4 2XU / Scotland