The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.

Operation method

Dephosphorylation of DNA fragments with alkaline phosphatase

Materials and Instruments

Bovine Small Intestine Alkaline Phosphatase Shrimp Alkaline Phosphatase Protease K Restriction Endonuclease DNA Samples
Chloroform EDTA Ethanol Phenol Chloroform SDS Sodium Acetate TE Tris-Cl CIP Dephosphorylation Buffer SAP Dephosphorylation Buffer
Water bath or heating plate

Move

I. Materials

1. Buffers and solutions

Chloroform

EDTA ( 0.5 mol/L, pH 8.0) or EGTA ( 0.5 mol/L, pH 8.0) (if using CIP)

Ethanol

Phenol: chloroform (1:1, V/V)

SDS (10% m/V) ( if using CIP)

Sodium acetate (3 mol/L, pH 7.0 [ if using CIP] and pH 5.2)

TE (pH 7.6)

Tris-Cl (1 mol/L, pH 8.5)

2. Enzyme and buffer

Bovine small intestinal alkaline phosphatase (CIP) or shrimp alkaline phosphatase (SAP) (US Biochemicals, Boehringer Mannheim, or Worthington Biochemicals)

10X CIP dephosphorylation buffer or 10X SAP dephosphorylation buffer

Protease K

Restriction endonuclease

3. Nucleic acids and oligonucleotides

DNA samples (0.1 to 10 μg [ 1-100 pmol])

4. Specialized equipment

Water bath or heating plate pre-set to 56°C, 65°C or 75°C (CIP) or 70°C (SAP)



II. Methods

1. Completely digest 1~10 μg (10~100 pmol) of DNA for dephosphorylation with the restriction endonuclease of choice.

2. Dephosphorylate the 5' end of restriction digested DNA with CIP or SAP.

(1) Dephosphorylation of DNA by CIP

① Add to the DNA:

10X CIP dephosphorylation buffer, 5 μl.

Water Add to 48 μl

② Add appropriate amount of CIP.

③ After incubating the reaction at 37℃ for 30 min, add the second aliquot of CIP and continue to incubate for 30 min.

After the incubation reaction, add SDS and EDTA (pH 8.0) to a final concentration of 0.5% and 5 mmoI/L, respectively, to inactivate CIP, and mix thoroughly to prepare the reagent, and then add Proteinase K to a final concentration of 100 μg/ml, and incubate for 30 min at 56 ℃.

⑤ Cool the reaction to room temperature and purify the DNA by extracting with phenol and chloroform twice, and then extracting with chloroform alone once.

(2) DNA dephosphorylation with SAP

① Add to the DNA:

10X SAP dephosphorylation buffer 5 μl

Water Add to 48 μl

② Add appropriate amount of SAP.

③ Incubate the reaction for 1 h at 37℃.

④ To inactivate SAP, transfer the reaction to 70℃ and heat for 20 minutes, then cool to room temperature.

3. Transfer the aqueous phase to a clean microcentrifuge tube and recover the DNA by standard ethanol precipitation in the presence of 0.1 v/v of 3 mol/L sodium acetate (pH 5.2) for SAP or 0.1 v/v of 3 mol/L sodium acetate (pH 7.0) for CIP.

4. Dry the precipitate at room temperature and dissolve in TE (pH 7.6) to a concentration of DNA > 2 nmol/ml.

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