Engineering BioBrick vectors from BioBrick parts/Dephosphorylation: Difference between revisions
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*Deionized, sterile H<sub>2</sub>O | *Deionized, sterile H<sub>2</sub>O | ||
*Linearized destination vector from [[Engineering BioBrick vectors from BioBrick parts/Restriction digest | restriction digest]] | *Linearized destination vector from [[Engineering BioBrick vectors from BioBrick parts/Restriction digest | restriction digest]] | ||
*Sterile 0.6mL plastic tubes | |||
==Equipment== | ==Equipment== | ||
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==Procedure== | ==Procedure== | ||
Vortex all reagents before use. | |||
#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> | #Add Antarctic Phosphatase. <br>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. | ||
Latest revision as of 08:42, 18 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
- Sterile 0.6mL plastic tubes
Equipment
- DNA Engine Peltier Thermal Cycler (PTC-200) from MJ Research, Inc. (now Bio-Rad Laboratories, Inc., Hercules, CA).
Procedure
Vortex all reagents before use.
- Add Antarctic Phosphatase buffer to a final concentration of 1X to linearized vector sample.
- Add Antarctic Phosphatase.
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.
