This protocol (provided by Andereson of the University of Texas Southwestern Medical Center) describes the introduction of restriction endonuclease sites at both the 5' and 3' ends of the cloned mammalian cDNA, and in some cases at the end of the 5' end of the cDNA, the change of a codon to the preferred codon of E. coli. This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Genetic engineering with PCR
Principle
This protocol (provided by Andereson at the University of Texas Southwestern Medical Center) describes the introduction of restriction endonuclease sites at both the 5' and 3' ends of a cloned mammalian cDNA, sometimes at the 5' end of this cDNA, changing some of the codons to E. coli's preferred codons, etc.
Materials and Instruments
Restriction endonuclease Heat-stable DNA polymerase Positive control DNA Template DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
Amplification buffer dNTP storage solution
Agarose or polyacrylamide gel Shielded tip Tubes or microtitre plates Positive displacement pipettes PCR instruments
1. Buffers and solutions
10X amplification buffer
4 dNTP storage solutions (20 mmol/L, pH 8.0)
2. Enzyme and buffer
Suitable restriction endonucleases
Heat-stable DNA polymerase
3. gels
Agarose gel or polyacrylamide gel
4. Nucleotides and oligonucleotides
Oligonucleotide primer 1 and primer 2 were dissolved in TE (10 μmol/L, pH 8.0).
Positive control DNA
Template DNA
5. Carrier
Plasmid DNA should be digested with the appropriate restriction endonuclease and purified by gel electrophoresis.
6. Specialized equipment
Shielded tips for automated micropipettes
Centrifuge tubes (0.5 ml, for thin-walled amplification reactions) or microtitre plates
Positive displacement pipettes
PCR instrument
II.
1. Design and synthesis of oligonucleotide primers for expected end modifications.
For example, the 5' primer designed according to the cDNA template was 5' dATCATATATGGCTCTG GATGAACT- GTGCCTGCTGGGACATGCT3', and the 3' primer was 5' dATAAGCTTTTATTAAGACA-GACTCAGCTCATGGGGGAGGCAA3', which introduced the Nde I (CATATG) site into the cDNA at the 5' end. Nde I (CATATG) site was introduced at the 5' end of the cDNA, which changed part of the codon of the cDNA to an E. coli favorite codon. The underlined nucleotides indicate that the oligonucleotide primers are different from the cDNA template. There is no precise determination of the number of base pairs required at the 3' end of an oligonucleotide primer to successfully amplify a target gene; however, it has been found that 8-10 pairs of base pairs are generally sufficient in specific work.
2. Add the components to the wells of a 0.5 ml sterilized centrifuge tube, amplification tube or sterilized titration plate in the following order and mix:
100 ng template DNA 10 μl
10X Amplification Buffer 5 μl
20 mmol/L 4 dNTP mix 5 μl
10 μmol/L Primer 1 (50 pmol) 5 μl
10 μmol/L Primer 2 (50 pmol) 5 μl
1~2 units of heat-stable DNA polymerase 1 μl
H2O make up to 50 μl
Set up two control reaction tubes and add all the above reagents except the template DNA. In the other reaction, add all the above reagents including the template DNA. Positive results are expected by PCR. The test tubes are used together with the control tubes for all subsequent experimental steps.
3. If the PCR instrument is not equipped with a heating cap, a drop of mineral oil (approx. 50 μl) should be added to the upper layer of the reaction mixture to prevent evaporation of the sample during the multiple heating and cooling cycles of the PCR reaction. If a hot-start PCR program is applied, add a layer of paraffin oil to the top of the reaction mixture. Place the centrifuge tube and microtiter plate on the PCR instrument.
4. Perform PCR amplification as follows. Typical procedures are denaturation, denaturation and polymerization (extension reaction); the corresponding cycling conditions and temperatures are listed below: 
5. 5% to 10% of each amplified sample is taken and analyzed by agarose gel electrophoresis or polyacrylamide gel electrophoresis, and a DNA marker is used to determine the size of the amplified fragments. The gels were generally stained with EB (ethidium bromide) or SYBR gold particles to observe the amount of amplification and fragment size.
-A successful amplification reaction should produce DNA fragments of the size we expect. Amplified bands can be identified by DNA sequence analysis, SoutKem hybridization and restriction endonuclease profiling.
6. If mineral oil was used to cover the top layer of sample liquid in the microcentrifuge tube (step 3), it can be removed by 150 μl of chloroform extraction at the end of the reaction.
7. For subsequent cloning, the DNA amplified fragments should be digested with the appropriate restriction endonuclease (the cleavage site is introduced at the 5' end of the primer, as in the example above with Nde I and Hind III). The digested fragments are purified by gel electrophoresis or ultrafiltration.
8. Establish an appropriate ligation reaction with the desired vector DNA, with a 3:1 molar ratio of insert to vector in the ligation reaction.