The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Experimenting with connectors on protruding ends
Materials and Instruments
T4 Phage DNA Ligase Polynucleotide Kinase Restriction Endonuclease Move I. MATERIALS For more product details, please visit Aladdin Scientific website.
ATP Ethanol Junction kinase buffer Phenol Chloroform Sodium acetate TE
Chromatography equipment Water bath
1. Buffers and solutions
ATP (10 mmol/L), ethanol, 10x junction kinase buffer, phenol: chloroform (1:1, V/V), sodium acetate (3 mol/L, pH 5.2), TE ( pH 8.0).
2. Enzymes and buffers
T4 phage DNA ligase, polyribonucleotide kinase, restriction endonuclease.
3. Nucleic acids and oligonucleotides
exogenous Nucleic acids and oligonucleotides
Exogenous or target DNA fragments, synthetic oligonucleotides or junctions, solubilized in TE (pH 8.0) to a final concentration of approximately 400 ug/ml. 4. Specialized equipment
Chromatography equipment, water bath with adjustable temperature up to 65 °C.
Methods
1. Phosphorylation of the junctions is performed in a sterile centrifuge tube:
Synthetic oligonucleotides or junctions are dissolved in 0.5-2.0 ug TE (pH 8.0), 1.0 ul of 10x junction kinase buffer, 1.0 ul of 10 mmol/L ATP, 10 ul of H2O, and 1.0 unit of T4 phage DNA ligase, and the system is incubated at 37°C for 1 h.
There is no need to passivate the phosphorylated junctions. It is not necessary to passivate the phosphorylated junctions because the ligation reaction can be carried out directly in the reaction mixture.
2. The reaction system for ligating the phosphorylated junctions to the replicated DNA fragments is as follows:
DNA fragments 100-200 ng, phosphorylated junctions molarized more than 10-20-fold, 10x ligase buffer 1.0 ul, T4 phage DNA ligase 0.1 unit of Weiss, 10 mmol/L ATP 1.0 unit, 10 mmol/L ATP 1.0 unit, 10 mmol/L ATP 1.0 unit. 10 mmol/L ATP 1.0 ul, H2O to 10 ul, and place the ligation mixture at 4°C for 6-16 h.
For maximum efficiency of ligation, the smaller the reaction system, the better, generally 5-10 μl. ATP is used as a component of the 10X ligation buffer in the reaction mixture to provide more space for the volume of the vector and the exogenous DNA fragments. Some manufacturers' ligation buffers already contain ATP, so it is not necessary to add ATP for application.
3. Incubate the ligation mixture at 65°C for 15 min to inactivate the DNA ligase.
4. Dilute the ligation product with 10 ul of the appropriate 10x restriction buffer. Add distilled water so that there are 50-100 units of restriction enzyme in a final volume of 100 ul.
5. Incubate the reaction system at 37°C for 1-3 h.
Restriction enzymes remove the polymers from the ends of the DNA fragments and form protruding ends. A large number of junctions are present in the reaction system and require digestion with a large amount of restriction enzyme.
6. The reaction mixture is extracted once with phenol: chloroform and the DNA is recovered by ethanol precipitation.
7. The DNA precipitate is collected by centrifugation at maximum speed for 15 min at 4°C in a microcentrifuge and re-dissolved in 50 ul of TE (pH 8.0).
8. The DNA solution obtained is passed over a column to remove excess junctions and enzymes. remove excessive amounts of junctions and digested fragments.
9. The modified DNA fragments can now be used to ligate to plasmid DNA with protruding ends.