Construction of recombinant DNA by polymerase chain reaction
Construction of recombinant DNA by polymerase chain reaction
The polymerase chain reaction constructs recombinant DNA for a wide range of uses (1) for sequencing primers (2) targeted mutagenesis (3) nucleic acid hybridization probes (4) PCR primers.
Operation method
basic program
Principle
Using the polymerase chain reaction (PCR), any two DNA fragments can be joined to form a new recombinant DNA molecule. By means of additional non-homologous nucleotides introduced in the PCR primers, the desired reading frame or cleavage site can be generated at the junction.
Materials and Instruments
DNA Move 1. Prepare template DNA, if the DNA is not purified by cesium chloride gradient, boil at 100°C for 10 min to inactivate nuclease.2. prepare oligonucleotide primers, and phosphorylate the hydroxyl group at the 5' end of the primer if the PCR product is to be cloned flat-end. Caveat 1. If non-specific bands are present, it is possible： （1） Inappropriate primer design, hairpin structure or dimerization;(2) Inappropriate annealing temperature； （3） The quality of polymerase is not good； （4） There is contamination； （5） Impure or excessive primers； （6） Excessive template. Common Problems With the continuous development of science and technology, new technologies including reverse transcription PCR, real-time quantitative PCR and other new technologies, as well as improved technologies such as multiplex PCR, DNAshuffing, PCR chip and solid-phase PCR, have been developed on the basis of traditional PCR technology. Currently there is the use of thermostatic rapid amplification technology as a DNA methylation-specific detection method to verify the genes of cancer, providing new methods and new ideas for in-depth exploration in the field of medicine. For more product details, please visit Aladdin Scientific website.
TE Anhydrous Ethanol DNA Polymerase CTP
Electrophoresis machine Centrifuge PCR machine
Figure I. Introduction of a single enzymatic microsite and generation of fusion proteins conforming to the reading frame by PCR reaction.3. Establish a standard amplification reaction by covering the surface with mineral oil and performing 20 to 25 rounds of amplification cycles under the following conditions: denaturation at 94°C for 1 min, annealing at 50°C for 1 min, extension at 72°C for 3 min, and a final cycle of extension at 72°C for 10 min as long as possible to keep the amplification product intact.
Figure 2, sequential PCR to construct recombinant DNA molecules, primer 1b and gene 2 homologous region; primer 1c has a homologous region with gene 14. Take a small amount of reaction mixture (4~8 ul) and test the amplification effect by agarose gel electrophoresis or polyacrylamide gel electrophoresis.5. Aspirate off the upper layer of mineral oil, extract with chloroform once to remove the residual mineral oil, extract with saturated phenol once, and then precipitate the DNA with anhydrous ethanol.
Figure 3. Insertion of enzymatic sites by reverse PCR
6. Centrifuge at high speed for 10 min at 4°C, and dissolve the precipitate with 20 ul TE buffer.
7. Further purification of PCR products with glass beads, electroshedding or phenol extraction with low-melting dispensing gel removes unincorporated dNTP primers, extraneous PCR products and template DNA.8. For cloning by flat ligation: Flatten the 3' end of the amplified fragment with DNA polymerase I (or klenowase).9. For cloning by sticky end ligation: Digest half of the PCR-only product in a 20 ul volume with a suitable enzyme in excess for several hours.10. Digest 0.2 to 2 ug of vector DNA in a 20 ul volume with a suitable end-complementary enzyme and de-glutamine with CIP to stop vector self-ligation.
11. Separate linearized vector by loose electrophoresis with normal agarose or low melting point agar.
12. Recover linearized vectors by glass beads, electroelution, or phenol extraction.
13. Ligate PCR fragments and linearized vectors.14. Take a portion of the ligation product and transform B. biglobosa, and extract plasmid DNA from the transformant pool by the small volume preparation method.
15. Digest the plasmid DNA of the transformants with suitable enzymes and analyze by agarose gel electrophoresis to confirm the insertion of the fragment. 16.
16. Determine the sequence of the amplified fragments in the plasmid DNA and check for mutations or screen the transformants for biochemistry or genetics.
Literature: Liao Ping, Liu Chazhen, Comparison of recombinant enzyme-mediated amplification technology and traditional polymerase chain reaction technology in the detection of DNA methylation in thyroid cancer, Chinese Medicine, 2003,8(6):797-799.