Dephosphorylation of linearised plasmid DNA for ligation

Dephosphorylation of linearised plasmid DNA for ligation.

Dephosphorylation of linearised plasmids is a method employed to prevent the majority of plasmids simply recircularising during the ligation step when cloning your DNA of interest.

It is especially useful when both protruding terminii of the linearised plasmid have been cut with the same enzyme (sticky ends) but is also used in blunt-ended ligations. Note: After ligation and transformation, it is essential to perform a diagnostic experiment (e.g. colony PCR with suitable primers) to ensure that the clone you choose contains your DNA fragment of interest, that it is in the correct orientation and concatamers have not formed.

In this lab we use Calf intestinal phosphatase (CIP) which is kept in the cold room Rm103.

It is supplied with 10x buffer:

10 mM ZnCl2

10 mM MgCl2

100mM Tris.HCl (pH8.0)

1 unit of CIP is sufficient to remove 100pmoles of 5' terminal phosphate groups in 30mins at 37ûC. (2µg of linearised plasmid (5kb) contains 1.4 pmoles of 5' terminal phosphate groups)

A typical reaction would be:

Total vol. =100 µl

2-3 µg of vector in 20-30 µl ddH2O

10 µl 10x CIP buffer

1µl CIP(1u/µl)

upto 100 µl ddH2O

Incubate at 37ûC for 1hr.

Optional=(Add EDTA to 5mM (1µl of 0.5M EDTA pH8.0) and heat inactivate at 65ûC for 1hr.)

Extract the DNA using phenol:choloform or the QIAEX desalting protocol.