Engineering BioBrick vectors from BioBrick parts/Dephosphorylation
Revision as of 20:36, 17 March 2008 by Reshma P. Shetty (New page: 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 percent...)
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.
- 5 units Antarctic Phosphatase from NEB
- 10X Antarctic Phosphatase buffer
- Deionized, sterile H2O
- Linearized destination vector from restriction digest
- Add Antarctic Phosphatase buffer to a final concentration of 1X to linearized vector sample.
- Add Antarctic Phosphatase.
Vortex phosphatase before pipetting to ensure that it is well-mixed.
Also, the phosphatase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add the correct amount, just touch your tip to the surface of the liquid when pipetting.
- Add deionized
- 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.
- Heat-inactivate for 5 mins at 65°C.
- Proceed directly to ligation step.