Phosphatase treatment of linearized vector: Difference between revisions
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==Materials== | ==Materials== | ||
*Linear DNA from [[Restriction Digest]] (heat-inactivation of restriction enzymes is necessary but [[Purification of DNA | DNA purification]] is not). | *Linear DNA from [[Restriction Digest | restriction digest]] (heat-inactivation of restriction enzymes is necessary but [[Purification of DNA | DNA purification]] is not). | ||
*[[Antarctic Phosphatase]] | *[[Antarctic Phosphatase]] | ||
*10X Antarctic Phosphatase buffer | *10X Antarctic Phosphatase buffer | ||
Revision as of 07:13, 1 June 2005
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
- Linear DNA from restriction digest (heat-inactivation of restriction enzymes is necessary but DNA purification is not).
- Antarctic Phosphatase
- 10X Antarctic Phosphatase buffer
Procedure
- Add Antarctic Phosphatase buffer to a final concentration of 1X to linearized vector sample.
- Add 1μL Antarctic Phosphatase (probably should make final glycerol concentration less that 5%?)
- 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.
