Flat-end cloning of PCR products
After the target gene is amplified by PCR and the sample is purified and recovered as necessary, the next step is to use flat ends for ligation and cloning, which is also a common technique in molecular biology experiments, and phage T4 DNA polymerase and other phage DNA polymerase enzymes are generally used to make up for the flat ends of the amplified DNA fragments (Weiner 1993; Chuang et al. 1995). This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Flat-end cloning of PCR products
Principle
After the target gene is amplified by PCR and the sample is purified and recovered as necessary, the next step is to use flat ends for ligation cloning, which is also a routine procedure in molecular biology experiments, which generally utilizes phage T4 DNA polymerase to make up for the flat ends of the amplified DNA fragments (Weiner 1993; Chuang et al. 1995). 1992) found that the presence of excess restriction endonuclease in the reaction solution significantly increased the yield of recombinant plasmids during ligation incubation. The endonuclease acts to cleave circular and linear tandem junctions, and the internal cleavage site is generated by self-ligation of the plasmid molecule. This method requires that the target DNA molecule be ligated to the vector to eliminate its restriction sites. The process must also prevent endonuclease digestion of the recombinants produced by the ligation reaction. The net effect of continuous generation (or regeneration) of linear vector molecules per unit length in the ligation reaction will drive the equilibrium of the ligation reaction in a direction that favors ligation of the vector to the insertion fragment to produce recombinants.
Materials and Instruments
Phage T4 DNA ligase Phage T4 DNA polymerase Restriction endonuclease Closed-loop plasmid DNA Target gene DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
ATP dNTP solution KGB wide area buffer
Agarose gel Water bath
1. Buffers and solutions
10 mmol/L ATP
2 mmol/L dNTP solution (contains four dNTPs)
10X KGB wide-field buffer (1 mol/L potassium acetate, 250 mmol/L Tris-acetate ( pH 7.6), 100 mmol/L MgAc (containing tetrahydrate), 5 mmol/L β-mercaptoethanol, 100 μg/ml bovine serum albumin)
2. Enzyme and buffer
Phage T4 DNA ligase
Phage T4 DNA Polymerase
Restriction endonucleases for cloning
Restriction Endonucleases
3. gels
Agarose gel
4. nucleic acids and oligonucleotides
Closed-loop plasmid DNA ( 50 μg/ml)
Target gene DNA (25 μg/ml) and PCR amplification products
5. Special equipment
The water temperature of the water bath was preset at 22℃
II.
1. In a microcentrifuge tube, add the following reagents sequentially and mix well:
50 μg/ml closed loop plasmid vector 1 μl
25 μg/ml PCR amplification target DNA 8 μl
10X KGB Broad Buffer 2 μl
H2O (see note below) 5 μl
10 mmol/L ATP 1 μl
2 mmol/L dNTP 1 μl
Restriction endonuclease 2 units
T4 DNA polymerase 1 unit
T4 DNA ligase 3 units
Note: Adjust the final reaction volume to 20 μl of reaction system with H2O.
Set up a control reaction tube with all the above reagents except PCR amplified target DNA.
2. Centrifuge the ligation mixture in a water bath at 22°C for 4 hours.
3. 5 μl of the ligation mixture from each of the two tubes is diluted with 10 μl of H2O and transformed into antibiotic-resistant E. coli receptor bacteria. This transformed bacterial fluid was spread on a petri dish containing appropriate antibiotics and medium with IPTG and X-gal.
4. Count the number of colonies on the petri dish for the experimental and control tubes.
Pick a white single colony that may contain the target gene DNA insertion fragments for culture amplification, extract the plasmid DNA, use the plasmid polyclonal sites within the insertion of exogenous DNA fragments flanking the enzyme sites, using the corresponding restriction endonuclease for enzymatic identification; can also be identified by colony PCR.
5. Determine the size of the cloned DNA fragments by agarose gel electrophoresis (using the molecular weight of the appropriate DNA marker as a control).
6. Use DNA sequence analysis, restriction endonuclease mapping or Southern hybridization to further characterize the correctness of the cloned target gene DNA fragments.