Purpose is to remove the 5' phosphates of DNA fragments and vectors, the
former can then be re-phosphorylated with 32P-labelled ATP to prepare labeled
primer for primer extensions (for toe-printing) while the latter can be used to improve the efficiency of cloning fragments.
There are two types of alkaline phosphatase used in our laboratory. The first is bacterial alkaline
phosphatase (BAP) which is very hardy and reliable but has the disadvantage that
preparations are sometimes contaminated with an exonuclease that can ruin a sticky
ended vector for cloning purposes. By carrying out the digestion at 68°C and phenol
extracting immediately and multiple times, the contaminating exonuclease can be
suppressed and then destroyed -- or so you hope. For these reasons, BAP
is rarely used and then only for dephosphorylating fragments before
labeling for sequencing. Use the
cleaner calf intestinal alkaline phosphatase (CIP) for cloning purposes.
In both cases it
is critically important to remove all traces of phosphatase at the end of the reaction for
BAP use multiple (3-4) phenol chloroform extractions since even a trace of residual
phosphatase activity will ruin your subsequent experiment. One of the biggest
advantages of using CIP is that it is killed by heating to 70°C for 10 minutes.
