Phosphatase treatment of linearized vector

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Current revision (07:49, 20 March 2007) (view source)
 
Line 13: Line 13:
#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==

Current revision

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

Personal tools