Gene synthesis can be applied to (1) metabolic pathway synthesis, (2) gene network construction, and (3) vaccine design.

Operation method

Gene Synthesis Experiment

Principle

Gene synthesis is the artificial synthesis of double-stranded DNA molecules in vitro, as opposed to oligonucleotide synthesis: oligonucleotides are single-stranded, with the longest fragment synthesized being around 100 nt, whereas gene synthesis is the synthesis of double-stranded DNA molecules, with a range of lengths from 50 bp to 12 kb. Gene synthesis is the artificial synthesis of genes, and it is one of the means of obtaining genes, as opposed to obtaining genes from pre-existing organisms. Gene synthesis does not require a template as opposed to obtaining a gene from an existing organism, and is therefore not limited by the source of the gene.

Materials and Instruments

DNA
dNTP DAN polymerase Ethanol TE Restriction Endonuclease Buffer
Water Bath Incubator

Move

1. 1 μg of each of the two oligonucleotides was added to a microcentrifuge tube, water was added to 17 μl, and 2 μl of 10× sequencing enzyme buffer was added. 70 ℃ was heated for 5 min, and then kept warm for 5 min at a suitable annealing temperature, and 2 μl was set aside for later analysis.

2. Add 2 μl of 4 kinds of dNTP mixture and 10U sequencing enzyme, and incubate at 30℃ for 30min.

3. Inactivate DNA polymerase at 70℃ for 10 min, and take 2 μl for later analysis.

4. Add 10× restriction endonuclease reaction buffer, add water and 20--100 U of restriction endonuclease suitable for cloning to 100 μl. Digest for more than 2 h at the appropriate temperature.

5. Phenol extraction, add 100 μl of 4 mol/l ammonium acetate and 400 μl of anhydrous ethanol, precipitate at -70°C for 15 min, centrifuge for 5 min, resuspend the precipitate in 100 μl of TE buffer, precipitate again with ethanol, wash the precipitate with 95% ethanol, dry it, and dissolve it in 20 μl TE buffer, and take 2 μl of it for later analysis.

6. Analyze the confirmatory raw material, extension product and digest product by sieve agarose gel electrophoresis to estimate the amount of extended oligonucleotides, and then subclone them with appropriate vectors.

7. Sequencing of several suitable subclones.

Caveat

1. Please be sure to use new (non-contaminated) tips, Microtube, etc. for preparation and dispensing of reaction solution to avoid contamination as much as possible.

2. When preparing the reaction system, attention should be paid to the use of pipettes, all liquids should be added slowly to the bottom of the tube, not to the wall of the tube, and the mixing of all liquids should be carried out with an oscillator, to avoid the production of air bubbles.

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