M0290 FAQs for Alkaline Phosphatase, Calf Intestinal (CIP), Phosphatases and Sulfurylases, Intl, NEB
New England Biolabs
FAVORITE TOOLS
Enzyme Finder
Double Digest
PCR Selection
NEBcutter
REBASE
Alkaline Phosphatase, Calf Intestinal (CIP) FAQ

Q1: Which phosphatase (CIP, Antarctic or SAP) should I use?
Q2: The number of colonies that do not contain an insert seems high, how can I tell if the CIP worked?
Q3: Will CIP work in restriction enzyme NEBuffers?
Q4: Does the DNA need to be purified after the restriction digest, prior to CIP treatment?
Q5: Does the DNA need to be purified after CIP treatment?
Q6: Will CIP dephosphorylate proteins?
Q7: Can Antarctic Phosphatase remove the 3' phosphate from DNA?
Q8: Can CIP be heat inactivated?
Q9: Why do some protocols recommend using CIP at 50C and some recommend 37C?

Q1: Which phosphatase (CIP, Antarctic or SAP) should I use?

A1: Antarctic Phosphatase (
NEB #M0289) is often a superior choice because it is 100% heat inactivated in 5 minutes at 65C; as a result you can proceed directly to the ligation reaction without further purification of vector DNA. Since neither CIP or Shrimp Alkaline Phosphatase (SAP) can be completely inactivated at 65C in 30 minutes, it may be necessary to purify the DNA after the phosphatase reaction. It is important to inactivate the phosphatase because residual active phosphatase can cause failure in subsequent ligation/transformation experiments.


Q2: The number of colonies that do not contain an insert seems high, how can I tell if the CIP worked?

A2: CIP is a very robust enzyme and it is rare for dephosphorylation not to go essentially to completion. Low numbers of colonies containing vector with insert may be the result of incomplete inactivation or removal of CIP, causing dephosphorylation of the insert during ligation. Ligation conditions should also be checked (see T4 DNA Ligase). Background may also be the result of uncut vector.

The following controls can be used to diagnose this problem:
1. Uncut vector: to check cell competency and antibiotic resistance.
2. Vector cut and not ligated: to check for uncut vector, gel purification may be required to remove the last 0.1% of uncut vector.
3. Vector cut and ligated: to check for intact ends and ligation conditions.
4. Vector cut, CIPed and ligated: to check for dephosphorylation, should be 3-5% of the vector cut and ligated.


Q3: Will CIP work in restriction enzyme NEBuffers?

A3: CIP will work in NEBuffers 2, 3, or 4, as well as the unique NEBuffers for, BamHI and EcoRI. NEBuffer 3 gives optimum activity. Add 2X more CIP if the buffer contains less than 50 mM salt.


Q4: Does the DNA need to be purified after the restriction digest, prior to CIP treatment?

A4: In most cases, CIP will work well in the reaction containing the restriction enzyme. Heat inactivate the restriction enzyme if possible. Some enzymes may bind tightly to the DNA ends and compete with CIP. Check the restriction enzyme data card for information regarding excessive DNA binding.


Q5: Does the DNA need to be purified after CIP treatment?

A5: Yes. Purify DNA by gel purification, spin-column purification or phenol extraction. CIP binds DNA tightly and is difficult to completely remove. To improve DNA purification do not use more CIP than recommended.


Q6: Will CIP dephosphorylate proteins?

A6: Yes. For more information see
Protocol for Dephosphorylating Proteins with CIP.


Q7: Can Antarctic Phosphatase remove the 3' phosphate from DNA?

A7: Yes. Antarctic Phosphatase is able to remove 3' phosphates from DNA.


Q8: Can CIP be heat inactivated?

A8: Not completely.


Q9: Why do some protocols recommend using CIP at 50C and some recommend 37C?

A9: It has been shown that DNA fragments with 3 extensions or blunt ends are slightly more difficult to dephosphorylate with CIP than those fragments with 5 extensions. CIP works slightly better on 3 extensions and blunt-ends at 50C. We have found that the difference in efficiency is about 20-25%. Some protocols also recommend the addition of more CIP for these difficult ends. For the sake of simplicity, we recommend 37C and an enzyme concentration of 0.5 unit/g in a 10 l reaction for all types of ends.