DNA cloning refers to the process of molecular manipulation in which a DNA fragment containing a target gene or other meaningful DNA is ligated in vitro with a vector DNA that is capable of self-replication, and then transferred into a host cell or a recipient organism for expression or further research, so DNA cloning is also known as molecular cloning, genetic manipulation or recombinant DNA technology.
Operation method
basic program
Materials and Instruments
DNA Move 1. Subcloning of the target DNA fragment encoding the protein downstream of the SP6 or T7 promoter of the plasmid vector.2. Preparation of plasmid DNA by CsCl/ethidium bromide centrifugation or PEG precipitation.3. Cut the DNA with a restriction endonuclease whose site is immediately downstream of the stop codon (ideally 50 to 200 bases) and is not present in the protein coding region. take a small sample and examine it in agarose gel electrophoresis. For more product details, please visit Aladdin Scientific website.
TE Nucleoside Triphosphate Mix RNA Polymerase Buffer Isobutanol NaOH TCA
Centrifuge Shaker
4. phenol extraction and ethanol precipitation were used to purify the DNA, which was redissolved in 50 μl TE buffer.5. Create 25 μl of the following reaction mixture at room temperature (to avoid precipitation of the DNA template by spermine).8 μl H2O
5 μl DNA
5 μl 5×nucleoside triphosphate mixture
2.5 μl 10×SP6 or T7 RNA polymerase buffer
2.5 μl 10 mmol/l arginine
1 μl RNAsin
1 μl SP6/T7 RNA polymerase
Hold at 40°C for 60 min, if using T7 RNA polymerase reaction, without spermine, make up 2.5 μl of water.6. Equilibrate the phenol by adding 25 μl of buffer, shake well and extract immediately. Transfer the aqueous phase to a new microcentrifuge tube and extract twice with isobutanol. Add 6 μl 10 mol/l ammonium acetate and 70 μl ethanol to precipitate RNA and wash once with ethanol.
7. Resolve RNA in 24 μl TE buffer, add 6 μl 10 mol/l ammonium acetate and 70 μl ethanol, precipitate RNA and wash with ethanol once. Dissolve RNA precipitated in 10 μl TE buffer, RNA should be immediately transcribed in vitro or frozen and stored at -70℃.8. Add 1-10 μl of RNA to the in vitro translation kit according to the manufacturer's instructions. 15 μCi [ 35S ]-labeled methionine is added, and a typical reaction is carried out in 30 μl of Citrus aurantium at room temperature for 30-60 min. after the reaction is completed, it can be stored at 0-4 ℃ for 1 week.
9. take 1 μl of the product of the translation reaction and add it to 50 μl of 0.1 mol/l NaOH at 37 °C for 15 min add 1 ml of 10% TCA on ice for 15 min. the precipitate is collected on a glass fiber filter membrane and the bulk of the admixed [ 35S ] methionine is determined using a liquid flash counter.
10. 3 μl of the translation reaction product was taken and electrophoresed by single-question SDS-PAGE including protein molecular weight standards. The [ 35S ] proteins were visualized and radiolucent autoradiography was performed with EN3HANCE fluorescence autoradiography for 1-4 h. The [35S] proteins were then detected and color-coded.