Difference between revisions of "Phosphatase treatment of linearized vector"

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#Incubate 60 mins at 37&deg;C.  <br> This should be sufficient to remove 5' phosphates even from 5' recessed ends like those produced by Pst I.
 
#Incubate 60 mins at 37&deg;C.  <br> This should be sufficient to remove 5' phosphates even from 5' recessed ends like those produced by Pst I.
 
#Heat-inactivate for 5 mins at 65&deg;C.
 
#Heat-inactivate for 5 mins at 65&deg;C.
#Proceed directly to [[Ligation | ligation]] step.
+
#Proceed directly to [[DNA ligation | ligation]] step.
  
 
==Notes==
 
==Notes==

Latest revision as of 04:49, 20 March 2007

To minimize self-ligated vector in your transformation, treat your linearized vector with a phosphatase to remove the 5' phosphates necessary for ligation. This should improve the percentage of colonies with inserts.

Materials

Procedure

  1. Add Antarctic Phosphatase buffer to a final concentration of 1X to linearized vector sample.
  2. Add 1μL Antarctic Phosphatase (probably should make final glycerol concentration less that 5%?)
  3. Incubate 60 mins at 37°C.
    This should be sufficient to remove 5' phosphates even from 5' recessed ends like those produced by Pst I.
  4. Heat-inactivate for 5 mins at 65°C.
  5. Proceed directly to ligation step.

Notes

NEB's Antarctic Phosphatase