cDNA synthesis technology

After the mRNA of the target gene has been identified by in vitro cell-free translation, the first strand of cDNA can be synthesized in the steps described in this paper.

Operation method

cDNA synthesis technology

Materials and Instruments

RNA
α32PdNTP mRNA Methylmercury hydroxide β-mercaptoethanol RNase inhibitor Primer 01igo dT Tris-HCl MgCl2 KCl Reverse transcriptase EDTA Phenol-chloroform Ethanol Agarose Hepes DTT DNA polymerase Nuclease S1
SephadexG-100 Centrifugal Column Chromatography Centrifuge Tubes Constant Temperature Water Bath Electrophoresis Instrument Low Temperature Centrifuge

Move

Synthesis of the first strand of cDNA:1. Add 50 pmol/L of a kind of α32PdNTP and 1 mg/ml of mRNA 10 μl (10 μg),100m mol/L methylmercury hydroxide 1 μl in an Ependorf tube and react for l0 minutes at room temperature to denature the RNA.2. Add 100m mol/l β-mercaptoethanol 2μl and 10m mol/l RNase inhibitor 2μl, and leave it at room temperature for 5 minutes. β-mercaptoethanol has the effect of stabilizing reverse transcriptase activity.3. To the above reaction mixture, add lmg/ml of primer 01igo dT (12-18 polymers) 10 μl, 1 mol/l Tris.HCl (pH8.3) 5 μl, 11 mol/l, KCl7 μl, 250m mol/l, _MgCl22 μl, then add 20 m mol/l of each of the four dNTP 2.5 μl, reverse transcriptase 2 μl (40 units), and then add 2.5 μl of each of the four dNTP. l (40 units), replenished with water to 50 μl, mixed thoroughly, and reacted at 42 ℃ for 1~3 hours.4. Add 0.5 EDTA (pH8.0) 2μl to terminate the reaction, then add 150m mol／l NaOH 25μl, 65 ℃ reaction for 1 hour, or 37 ℃ for 8 hours, hydrolyze the mRNA template to get the first strand of cDNA, and then add 25μl of each of the pH8.0, 1m mol／l Tris.HCl, and neutralize its pH.5. Measure the radioactivity and calculate the amount of synthesized DNA. In practice, the yield of the first strand of cDNA often does not exceed 10% to 30% of the amount of poltAmRNA.6. The cDNA is extracted with an equal volume of phenol-chloroform, the aqueous phase is extracted and separated from the remaining dNTP by centrifugal column chromatography on a Sephadex G-100 column, and precipitated with 2.5 times the volume of 95% cold ethanol.7. The first strand of the synthesized cDNA was electrophoresed on a 1.4% alkaline agarose gel, and the molecular weight standard was the end-labeled pBR322 restriction fragment. After electrophoresis, X-rays were spread, and the size of the first strand of the cDNA was determined by autoradiography.Synthesis of the second strand of cDNA:1. Precipitate the first strand of cDNA by centrifugation and resuspend it in 50 μl of water, add 50 μl of 2× second strand buffer (0.2 mol/l Hepes, pH 6.9, 20 m mol/l MgCl₂, 5 m mol/l DTT, 0.14 mol/l KCl, 1 m mol/l 4 kinds of Dntp), and mix well.2. Add 20~50 units of E.coli DNA polymerase K1enow fragment to each microgram of substrate, and react at 15℃ for 20 hours, this enzyme can recognize the hairpin loop at the 3'end of cDNA, and use it as the primer, the first strand of cDNA as the template, and then start the synthesis of the second strand.3. Add 0.5 mol／l EDTA 2μl to terminate the reaction. Samples were electrophoresed and dsDNA was isolated and precipitated in the same way as the first strand. dsDNA synthesized in this way is not a full-length DNA molecule, so it needs to be synthesized further by reverse transcriptase.4. Dissolve the above dsDNA in 20 μl of water, then add: 1 mol/l Tris.HCl pH8.3) 5 μl, 1 mol/l KCl 7 μl, 250m mol/l MgCl₂ 2 μl, 20m mol/l 4 kinds of 'dNTP' each 2.5 μl, 700m mol/l β-MCE 2 μl. Add water to 48 μl, add reverse transcriptase 2 μl (40 units), and keep the reaction at 42℃ for 1 hour.5. Add 0.5 mol／l EDTA 2μl to terminate the reaction, take the sample for electrophoresis, extract in the same way, and isolate the precipitated dsDNA.6. Use the double-stranded cDNA and single-stranded cDNA synthesized in two times to electrophoresis on 1.4% alkaline agarose gel, and observe after autoradiography, if the length of the dsDNA fragment is twice as long as the length of the first stranded cDNA, then the synthesis is successful.7. Digest the dsDNA with nuclease S1 to remove the hairpin loop connecting the two strands.

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