The cloned PCR product is ligated into a T vector.
DNA fragments amplified by Taq DNA polymerase PCR with a 3' prominent A base are efficiently cloned into T vectors that have an unpaired 3' T base complementary to the A base (Holton and Graham 1991; Marchuk et al. 1991). This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
The cloned PCR product was ligated into the T vector
Principle
DNA fragments amplified by Taq DNA polymerase PCR with a 3' prominent A base are efficiently cloned into T vectors that have an unpaired 3' T base complementary to the A base (Holton and Graham 1991; Marchuk et al. 1991).
Materials and Instruments
Phage T4 DNA Ligase PCR Amplified Target DNA T Vector Move I. Materials For more product details, please visit Aladdin Scientific website.
Agarose gel Water bath
1. Enzyme and buffer
Phage T4 DNA ligase
2. gels
Agarose gel
3. nucleotides and oligonucleotides
PCR amplified target DNA ( 25 μg/ml)
4. carrier
T Carrier
5. Special equipment
Water bath with preset water temperature at 14°C
II. Methods
1. In a microcentrifuge tube, add the following connection mixture in sequence:
25 μg/ml amplified target sequence DNA 1 μl
T plasmid 20 ng
10X ligation buffer 1 μl
Phage T4 DNA Ligase 3 units
H2O make up to 10 μl
2. Incubate the ligand mixture at 14℃ for 4 hours.
3. 5 μl of each of the above two tubes was 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 the appropriate antibiotic and medium with IPTG and X-gal.
4. Count the number of colonies on the petri dish for the experimental and control groups. Pick the white colonies that may contain the target gene DNA insertion fragments for culture amplification, extract the plasmid DNA, use the plasmid polyclonal site insertion of exogenous DNA fragments flanking the enzyme sites, and use the corresponding restriction endonuclease for enzyme digestion and identification; or directly use the colony PCR for identification.
5. Identify the size of the cloned DNA fragments by agarose gel electrophoresis (using the appropriate molecular weight of the DNA marker 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 fragments.