When you dephosphorylate a vector for cloning, you remove the 5′ phosphate groups required by DNA ligase to join the phosphodiester DNA backbone together. This prevents your vector from ligating back to itself during the ligation step and decreases your background.

Various alkaline phosphatases exist, including calf intestinal phosphatase (CIP), Shrimp and Antarctic phosphatases. The most commonly used phosphatase is CIP, but it is difficult to heat inactivate. For this reason, new phosphatases, such as Antarctic phosphatase, were developed that can be heat inactivated.

But what if you just want to stick with CIP? Here are a couple of protocols you can use.

DNA Dephosphorylation—Protocol 1

Use for dephosphorylating DNA in a restriction digestion reaction/solution

50-100 µL of DNA (5µg) in a restriction digest reaction/solution
1-2 µL of CIP enzyme (1 unit/µL)

  • Add 1-2 µL of CIP directly to your restriction digest. CIP is stable and active in most restriction digestion buffers. For example, NEB CIP works in all 4 NEB buffers. If you haven’t inactivated the restriction enzymes, be careful of STAR activity caused by excessive glycerol.
  • Incubate the sample for 30-60 minutes at 37°C.
  • Inactivate/remove the CIP as described in Tip 1.

DNA Dephosphorylation—Protocol 2

Use for dephosphorylating DNA in TE or H2O

20-40 µL of DNA (5 µg) in TE or H2O
5 µL of 10x CIP buffer
1-2 µL of CIP enzyme (1unit/µl)
Make up to 50 µL with water

  • In a sterile 1.5 mL microfuge tube, add the DNA, then the CIP buffer and then the 1-2 µL of CIP.
  • Mix thoroughly with a pipette tip and incubate for 30-60 minutes at 37°C.
  • Inactivate/remove the CIP as described in Tip 1.

Tip for Using Calf Intestinal Phosphatase

Tip 1:  Inactivation/Removal of CIP

The following three methods are available for inactivating or removing CIP:

Heat Inactivation

  • Note the MgCl2 concentration in the reaction and add EDTA (pH 8.0) to an equal final concentration.
  • Incubate the reaction at 65°C for 15 min.

Organic Extraction

  • Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1).
  • Vortex thoroughly and centrifuge at 14,000 × g at room temperature for 5 min.
  • Carefully remove the upper, aqueous phase and transfer it to a fresh microcentrifuge tube.
  • Add 0.1 volume of 3 M sodium acetate and vortex.
  • Add 2.5 volumes of 100% EtOH. (Note: Do not substitute NH4OAc for NaOAc because NH4 ions inhibit T4 polynucleotide kinase.)
  • Vortex the mixture thoroughly and centrifuge at 14,000 × g at room temperature for 5 min.

Gel Purification

  • Run the reaction on an agarose gel and cut the appropriate band out of the gel.
  • Use a standard gel purification kit to isolate the DNA.
  • Tip 2: You Need Phosphate Groups

Make sure the fragment you are going to ligate into the dephosphorylated vector possesses 5’phosphate groups. Standard oligos/primers and PCR products are generally not phosphorylated. You must treat them with T4 polynucleotide kinase prior to ligation. Alternatively, add restriction sites to the ends of the PCR product and digest the DNA prior to ligation.

Tip 3: Don’t Let it Sink

CIP is stored in a glycerol buffer for stability, but this means it sinks to the bottom of aqueous solutions. When you add the CIP, watch it drop into the DNA mixture by doing it with the tube held up in front of you, and then ensure it is properly re-suspended before incubation.

Tip 4:  Don’t Use too Much

CIP binds DNA tightly and is difficult to completely remove. To improve DNA purification do not use more CIP than recommended.

Make sure you follow these protocols to use calf intestinal phosphatase correctly and increase your cloning efficiency.