Removal of the phosphate group at the 5' end prevents self-ligation and cyclization of plasmid DNA. In an in vitro ligation reaction, DNA ligase forms a phosphodiester bond between two neighboring molecules. However, this reaction can only be completed if one molecule has a phosphate group at the 5' end and the other has a hydroxyl group at the 3' end. This experiment is from "Molecular Cloning Laboratory Guide, Third Edition", translated by Huang Peitang et al.
Operation method
Dephosphorylation assay of plasmid DNA
Principle
Removal of the phosphate group at the 5' end prevents self-ligation and cyclization of plasmid DNA. In an in vitro ligation reaction, DNA ligase forms a phosphodiester bond between two neighboring molecules. However, this reaction can only be completed if one molecule has a phosphate group at the 5' end and the other has a hydroxyl group at the 3' end.
Materials and Instruments
Calf Intestine Alkaline Phosphatase (CIP) or Shrimp Alkaline Phosphatase (SAP) Proteinase K Restriction Endonuclease Carrier DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
EDTA EGTA Ethanol Phenol Chloroform SDS TE Tris-Cl
Agarose gel Water bath
1. Buffers and solutions
EDTA ( 0.5 mol/L, pH 8.0), EGTA ( 0.5 mol/L, pH 8.0), Ethanol, Phenol, Phenol:Chloroform (1:1, V/V), SDS (10%, m/V), Sodium Acetate (3 mol/L pH 5.2 and pH 7.0), TE ( pH 8.0), Tris-Cl ( 10 mmol/L, pH 8.3) .
2. Enzyme and buffer
Calf intestinal alkaline phosphatase (CIP) or shrimp alkaline phosphatase (SAP), proteinase K ( 10 mg/ml), restriction endonuclease.
3. Gel
Agarose gels (0.7%) were prepared with TBE containing 0.5 μg/ml ethidium bromide.
4. nucleic acids and oligonucleotides
Vector DNA ( closed loop plasmid).
5. Specialized equipment
Water bath adjustable to 56℃ or 65℃.
Methods
1. Digest a reasonable amount (10 μg) of closed-loop plasmid DNA for 1 h with 2-3 times excess of restriction enzyme.
2. Remove a small portion (0.1 μg) of DNA and electrophoresis it on an agarose gel (0.7%) containing ethidium bromide to check the degree of digestion, and use the undigested plasmid DNA as a control. If digestion is incomplete, add more enzyme and continue digestion.
3. If digestion is complete, extract the DNA once with phenol: chloroform and precipitate with ethanol to recover the DNA. place the ethanol solution on ice for 15 min.
4. Recover the DNA by centrifugation at maximal speed at 4°C. Dissolve the DNA with 110 μl of 10 mmol/L Tris-Cl (pH 8.3).
Reserve 20 μl of DNA for later use as a control.
5. Add 10 μl of 10X CIP or 10X SAP buffer and an appropriate amount of calf intestine alkaline phosphatase (CIP) or shrimp alkaline phosphatase (SAP) to the remaining 90 μl of linear DNA and incubate as described in Table 1-8. 
6. Inactivate the phosphatase:
Inactivate CIP: After the incubation time, add SDS and EDTA (pH 8.0) to a final concentration of 5% and 5 mmol/L, respectively, mix thoroughly, and then add Proteinase K to a final concentration of 100 μg/ml, and incubate for 30 min at 55 ℃. CIP can also be inactivated by incubation at 65 °C for 30 min (or 75 °C for 10 min) in the presence of 5 mmol/L EDTA 10 mmol/L EGTA (pH 8.0).
or
Inactivation of SAP: incubate the reaction mixture in dephosphorylation buffer at 65 °C for 15 min.
At the end of the dephosphorylation reaction, it is important to remove or completely inactivate alkaline phosphatase before proceeding with the ligation reaction. Although either CIP or SAP can be inactivated by the methods described above, we recommend that the dephosphorylation reaction mixture be extracted with phenol:chloroform prior to ligation with dephosphorylated DNA.
7. Allow the reaction mixture to cool to room temperature, then extract once with phenol and once again with phenol:chloroform.
8. recover the DNA by ethanol precipitation. mix the solution again and place on ice for 15 min.
9. Recover the DNA by centrifugation at maximum speed at 4°C. Wash the DNA precipitate with 70% ethanol and centrifuge again at 4°C.
10. Carefully discard the supernatant and place the open tube on the bench until the ethanol has evaporated.
11. Dissolve the precipitated DNA in TE (pH 8.0) to a concentration of 100 μg/ml. store the DNA in small portions at -20 °C. 