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Alkaline phosphatase from
the psychrophilic strain Shewanella sp.
SIB1 (PAP) has both merits of BAP and CIAP.
Alkaline Phosphatase from psychrophilic bacterium
Alkaline phosphatase from the psychrophilic strain Shewanella sp. SIB1 (PAP) has both merits of BAP and CIAP.
Alkaline phosphatase from the psychrophilic strain Shewanella sp. SIB1 (PAP) can easily be heat inactivated as SAP and CIAP. On the other hand, BAP can not be easily inactivated even by phenol extraction. Refer to this place
Because activity of the enzyme at 60°C is about four times that at 37°C, the enzyme can easily removes phosphate from not only protruding 3'-end but also blunt end or recessed 5'-end at 60°C for 30min. (On the other hand, SAP and CIAP were rapidly inactivated at 60°C.)
Alkaline Phosphatase (PAP)
from Shewanella.sp. SIB1
Advantage of PAP
Although BAP is hard to be inactivated, PAP is easily inactivated by heat.
As shown in figure below, PAP treatment produces a larger number of white colonies on plates than BAP treatment after transformation. Because active BAP survives after inactivation treatments, active BAP remove phosphate of insert DNA during the ligation reaction and result in the decrease the ligation efficiency. The experiment shows that PAP is easily inactivated before ligation but not BAP. In order to get sufficient amount of white colony after BAP treatment, insert DNA must be added at high insert: vector ratio into ligaton reaction to overcome surviving BAP or more than two times of phenol extraction must be carried out.
One μg of the EcoRI cleaved pBluescript SK (+) vector was dephosphorylated by 0.5 unit of BAP or 5 unit PAP at 37°C. After dephosphorylation, the reaction mixtures were treated as follow,
No-treatment: The reaction mixtures were directly used for ligation reaction.
Phenol: Equal volume of phenol was added to the reaction mixture and it was vortexed for 30 seconds. Then the mixture was extracted by ether, and precipitated by ethanol. The precipitate was dried up and dissolved by dH2O. It was used for ligation reaction.
Heat: The reaction mixtures were just heat at 95°C for 5 min for PAP or at 100°C for 5 min for BAP. These were directly used for ligation reaction.
After above treatments, EcoRI cleaved, dephosphorylated pBluescript SK (+) vector were ligated to a 1kb insert DNA fragment. Competent XL1-Blues were transformed with the ligation products. Number of white colony was shown in figure.