Phosphatase treatment of linearized vector
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#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. | ||
| - | #Proceed directly to [[ | + | #Proceed directly to [[DNA ligation | ligation]] step. |
==Notes== | ==Notes== | ||
[http://www.neb.com/nebecomm/products/productM0289.asp NEB's Antarctic Phosphatase] | [http://www.neb.com/nebecomm/products/productM0289.asp NEB's Antarctic Phosphatase] | ||
| + | |||
| + | [[Category:Protocol]] | ||
| + | [[Category:DNA]] | ||
| + | [[Category:In vitro]] | ||
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
- 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.
