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Dephosphorylation of DNA ends with Calf Intestinal Alkaline Phosphatase (CIP)

(KA Longo, 1/01)
  1. After your DNA has been quantified, calculate the volume needed for 1 ug.
  2. Calculate the number of pmol ends per µg of your DNA. (Note: the DNA has been cut, so there will be two ends per DNA molecule.) Use the following equation:
  3. Determine the amount of CIP enzyme that will be needed for complete dephosphorylation using the following chart:
      CIP Units
    5' recessed termini1
    5' overhang termini 
    Blunt termini 0.01 

  4. Determine the conditions for the reaction using the following chart:
     Temp (C) Time
    5' recessed termini50 1 h
    5' overhang termini 37 30 min
    Blunt termini 50 1 h

  5. Mix the following:
     ddH2O---
     10x CIP buffer**5 µL 
    CIP* --- 
    cut DNA (1 µg) --- 
     TOTAL VOLUME50 µL

    *CIP will likely need to be diluted before it is added to the reaction. The dilution buffer is typically provided with the enzyme (25 mM Tris-HCl (pH 7.6), 1 mM MgCl2, 0.1 mM ZnCl2, 50% (v/v) glycerol).
    **The 10 x CIP buffer is identical to NEB Buffer 3, and CIP is also reasonably compatible with buffers 1, 2, and 4. Properly diluted CIP can be added straight into restriction digests, although results may vary appreciably.

  6. Run the reaction using the appropriate conditions
  7. CIP MUST be removed from the reaction if further cloning is to work. The simplest (and most effective) method is agarose gel extraction. Phenol chloroform extraction and ethonal/acetate precipitation can also be used, but residual phenol is hard to remove completely, and can inhibit ligations.