Promega CIAP Protocol

2015-01-13

azim58 - Promega CIAP Protocol

CIAP Promega Protocol

CIAP buffer last seen -20 #62 2nd shelf top left metal box row. 2nd box
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Note that this phosphatase reaction can be performed directly after a
restriction digest in the same restriction digest mixture and before a
gel purification.
Add 10X CIAP Reaction Buffer
Dilute sufficient CIAP for immediate use in CIAP 1X Reaction buffer to a
final concentration of 0.01 u/uL. Each picomole of DNA ends will require
0.01u CIAP. Andrey mentioned that you may want to add 10-20% extra.

* ex calculation
  114.4*ng*1*mol*"bp"/(660e9*ng)*6.02e23*"bp"/(1*mol*"bp")*1*"DNAmolecules"
  /(3296*"bp")*2*"DNAends"/(1*"DNAmolecules")*1*"mol"*"DNAends"/(6.02e23*"D
  NAends")*1e12*"pmol"*"DNAends"/(1*"mol"*"DNAends") = 0.105 DNAends pmol
  0.105+0.2*0.105 = 0.126
Incubate 37 C 15 min then 56 C 15 min.
Add another aliquot of CIAP.
Repeat incubation (Incubate 37 C 15 min then 56 C 15 min)
They recommend adding 300 uL of CIAP stop buffer. Andrey thinks that this
is unnecessary.
Ethanol precipitate the DNA or perform a gel purification. It is best to
ethanol precipitate the DNA if you are working with an already cut and
gel purified fragment. It is best to gel purify if this phosphatase
reaction was done immediately after a restriction digest.

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