The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Introduction of restriction endonuclease sites at the end of amplified DNA products by PCR amplification
Materials and Instruments
Phage T4 DNA ligase Restriction endonuclease Target DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
Chloroform EDTA Ethanol Phenol Chloroform Sodium acetate TE
Agarose gel Water bath
1. Buffers and solutions
Chloroform
EDTA ( 0.5 mol/L, pH 8.0)
Ethanol
Phenol: chloroform (1:1, V/V)
Sodium acetate (3 mol/L, pH 5.2)
TE ( pH 7.5)
2. Enzyme and buffer
Phage T4 DNA Ligase
restriction endonucleases
3. gels
Agarose gel
4. nucleic acids and oligonucleotides
Forward primer (20 μmol/L) and reverse primer (20 μmol/L) were dissolved in water.
Target DNA
5. carrier
Plasmid DNA should be digested with the appropriate restriction endonuclease and purified by gel electrophoresis.
6. Specialized equipment
Water bath
II. Methods
1. Amplify the target fragment by PCR reaction using forward and reverse primers designed in the material part of the protocol and divided into 4 identical 50 μl volume reaction tubes. Combine the reaction solutions of the 4 reaction tubes, the solution should contain 200~500 ng of the desired amplification product.
2. If the PCR product contains more than one or two amplified DNA bands by electrophoresis, the amplified fragment can be purified by low-melting-point agarose gel. If gel electrophoresis is not used for purification, the prepared PCR amplified DNA can be purified by phenol/chloroform extraction and ultrafiltration in a Cemricon-100 concentrator. The purified amplification product is dissolved in TE (pH 7.5) at a concentration of 25 μg/ml.
3. About 100 ng of the purified PCR product was digested in 20 μl of digestion system with 1.0-2.0 units of the appropriate restriction endonuclease. Incubate the reaction solution for 1 h at optimal temperature.
4. After digestion, the reaction mixture was adjusted to 10 μl with H2O and 0.5 mol/L EDTA was added to a final concentration of 0.5 mol/L. The reaction solution was extracted once with phenol, once with chloroform and once with chloroform.
5. Transfer the upper aqueous phase of the reaction solution to a new centrifuge tube, add 3 moI/L sodium acetate (pH 5.2) to a final concentration of 0.3 mol/L, add 2 times the volume of ethanol, and store at 0℃ for 30 min.
6. Place the tube in a microcentrifuge and centrifuge at high speed for 5 min at 4℃ to precipitate the DNA, discard the supernatant, then wash the precipitate with 70% ethanol, centrifuge the solution again, discard the supernatant, and allow the DNA precipitate to dry in the air for a few minutes.
7. Dissolve the DNA sample in 10 μl of water.
8. In a microcentrifuge tube, add the following reagents in sequence and mix:
25 μg/ml amplification target DNA 1.0 μl
Plasmid DNA 20 ng
10X ligation buffer 1.0 μl
T4 DNA Ligase 1 unit
Adjust the final reaction volume to 10 μl of reaction system with H2O.
Add ATP to a final concentration of 1 mmol/L if desired.
In the ligation reaction solution for targeted cloning, the molar ratio of purified target DNA to digested plasmid vector should be 1:1. Set up a control reaction tube with all of the above reagents except plasmid DNA.
9. Incubate the ligation mixture in a water bath at 16°C for 4 hours.
10. 5 μl of each of the above two tubes of ligand solution was diluted with 10 μl of water and transformed into antibiotic-resistant E. coli receptor bacteria. This transformed bacteria was spread on petri dishes containing suitable antibiotics and medium with IPTG and X-gal.
11. Count the number of colonies on the petri dish for the experimental and control tubes. Pick white colonies that may contain inserted fragments of the target gene DNA for culture amplification, extract the plasmid DNA, and identify it by digestion with the appropriate restriction endonuclease using enzymatic sites flanking the inserted exogenous DNA fragments within the plasmid polyclonal site, or by colony PCR.
The ratio of blue to white spots varied between 1:5 and 2:1 in different experiments.
12. Identify the size of the cloned DNA fragments by agarose gel electrophoresis (using the molecular weight of the appropriate DNA marker as a control).
13. The correctness of the cloned target gene DNA fragments is further characterized by DNA sequence analysis, restriction endonuclease mapping or Southern hybridization.