Engineering BioBrick vectors from BioBrick parts/Dephosphorylation: Difference between revisions
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#Add Antarctic Phosphatase buffer to a final concentration of 1X to linearized vector sample. | #Add Antarctic Phosphatase buffer to a final concentration of 1X to linearized vector sample. | ||
#Add Antarctic Phosphatase. <br>Vortex phosphatase before pipetting to ensure that it is well-mixed. <br>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 Antarctic Phosphatase. <br>Vortex phosphatase before pipetting to ensure that it is well-mixed. <br>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, sterile H<sub>2</sub>O | #Add deionized, sterile H<sub>2</sub>O. | ||
#Incubate 60 mins at 37°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°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°C. | #Heat-inactivate for 5 mins at 65°C. | ||
Revision as of 20:39, 17 March 2008
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
- 5 units Antarctic Phosphatase from NEB
- 10X Antarctic Phosphatase buffer
- Deionized, sterile H2O
- Linearized destination vector from restriction digest
Equipment
- DNA Engine Peltier Thermal Cycler (PTC-200) from MJ Research, Inc. (now Bio-Rad Laboratories, Inc., Hercules, CA).
Procedure
- 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, sterile H2O.
- 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.
