Molecules: A guide to blunt-end cloning of PCR products!
Let’s take a look at the principle first: the target gene is amplified by PCR, and after the necessary purification and recovery of the sample, blunt-end ligation cloning is also a conventional technical route used in molecular biology experiments. The experiment generally uses phage T4 DNA polymerase, etc. Fill in the ends of amplified DNA fragments (Weiner 1993; Chuang et al. 1995). Liu and Schwartz (1992) found that during the incubation process of the ligation reaction, the presence of excess restriction endonuclease in the reaction solution can significantly increase the yield of recombinant plasmid. This endonuclease cleaves circular and linear concatemers, and this internal cleavage site is generated by the self-ligation of plasmid molecules. This method requires that ligation of the target DNA molecule to the vector eliminates its restriction sites. During this process, it is also necessary to prevent endonucleases from digesting the recombinants produced by the ligation reaction. In the ligation reaction, the net effect of the continuous production (or regeneration) of linear vector molecules per unit length will drive the equilibrium state of the ligation reaction in a direction that is conducive to the ligation of the vector and the inserted fragment to produce recombinants.
Materials and instruments used in the process: phage T4 DNA ligase, phage T4 DNA polymerase, restriction endonuclease, closed-circle plasmid DNA, target gene DNA, ATP, dNTP solution, KGB wide area buffer; agarose gel , water bath box
The steps are:
1. Prepare materials
1. Buffers and solutions
10 mmol/L ATP
2 mmol/L dNTP solution (contains four dNTPs)
10X KGB wide area buffer (1 mol/L potassium acetate, 250 mmol/L Tris-acetate (pH 7.6), 100 mmol/L MgAc (contains tetrahydrate), 5 mmol/L β-mercaptoethanol, 100 μg/ ml bovine serum albumin)
2. Enzymes and buffers
Phage T4 DNA ligase
Phage T4 DNA polymerase
Restriction endonucleases for cloning
restriction endonuclease
3. Gel
agarose gel
4. Nucleic acids and oligonucleotides
Closed circular plasmid DNA (50 μg/ml)
Target gene DNA (25 μg/ml) and PCR amplification product
5. Special equipment
The preset water temperature of the water bath is 22℃
2. Operation steps and methods
1. In a microcentrifuge tube, add the following reagents in sequence and mix well:
50 μg/ml closed-circle plasmid vector 1 μl
25 μg/ml PCR amplification target DNA 8 μl
10X KGB wide area buffer 2 μl
H2O 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: Use H2O to adjust the final reaction volume to 20 μl reaction system.
Set up a control reaction tube and add all the above reagents except for PCR amplification of target DNA.
2. Incubate the centrifuge tube of the ligation reaction mixture in a 22°C water bath for 4 hours.
3. Take 5 μl of the ligation reaction mixture sample from the above two test tubes, dilute it with 10 μl H2O, and transform it into competent E. coli recipient bacteria with antibiotic resistance. Spread this transformed bacterial solution onto a plate containing appropriate antibiotics and media containing IPTG and X-gal.
4. Count the number of colonies on the petri dish between the experimental tube and the control tube.
Pick a white single colony that may contain the target gene DNA insert for culture and amplification, extract the plasmid DNA, and use the enzyme cutting sites flanking the inserted exogenous DNA fragment in the plasmid multiple cloning site to cut with the corresponding restriction endonucleases. Nuclease digestion and identification; colony PCR can also be used for identification.
5. Determine the size of the cloned DNA fragments by agarose gel electrophoresis (using an appropriate DNA marker molecular weight as a control).
6. Use DNA sequence analysis, restriction endonuclease mapping or Southern hybridization to further identify the correctness of the cloned target gene DNA fragment.
