1, preparation of denatured double-stranded template
① Take 1.5ml of logarithmic growth period culture broth (recombinant plasmid template containing virus nucleic acid to be detected), centrifuge to remove supernatant, resuspend the colony with 150ml tris/ glucose buffer, and leave it at room temperature for 5min.
② Add 300ml 1% SDS and 0.2mol/L NaOH, back mix for about 15 times, leave it at room temperature for 15 minutes, add 25 ml of 3 mol/L sodium acetate (pH4.5), back mix for about 15 times, and leave it in an ice bath for 45 minutes.
③ Transfer 650ml of supernatant to a new tube, add 650ml of isopropanol, mix well, stand at room temperature for 65438 00 minutes, centrifuge for 5 minutes, discard isopropanol, and vacuum dry the precipitate.
④ Dissolve DNA with 125ml TE(pH 8.0), add 375ml 4mol/L LiCl, ice bath for 20min, and centrifuge at 4℃ for 5min.
⑤ Transfer the supernatant to a new tube, extract with saturated phenol, then extract with chloroform, add 2 times of isopropanol, precipitate at room temperature for 30 minutes, centrifuge for 5 minutes, and discard the supernatant.
⑥ Wash the precipitate with ice-cold 70% ethanol, centrifuge for 5 minutes, discard the supernatant and dry it, and redissolve the precipitate with 50 ml te buffer. The content of plasmid DNA was determined by ultraviolet spectrophotometer.
⑦ Take 0.2mg plasmid DNA, adjust the volume to 9ml, add 1ml 2mol/L NaOH and 2mmol/L EDTA, leave it at room temperature for 5 minutes, and add 2ml 30mol/L sodium acetate (pH 4.5) and 8ml water.
⑧ Add 6ml of ice-cold absolute ethanol, mix and put in a dry ice/ethanol bath for 65438 05 minutes, centrifuge at 4℃ for 5 minutes, carefully discard the supernatant, wash the precipitate with ice-cold 70% ethanol, centrifuge for 5 minutes and carefully discard the supernatant, drain the precipitate in vacuum, and re-dissolve the precipitate with te buffer.
2. Extension and termination of the reaction
Take the dideoxy chain termination reaction using T7DNA polymerase as an example.
① Take four 0.5ml small centrifuge tubes, label them with G, A, T and C respectively, add 7ml denatured double-stranded DNA template (1 and 2mg respectively) to each tube, mix 1ml oligonucleotide primer with 2ml 5× sequencing buffer, keep the temperature at 65℃ for 2 minutes, and cool at room temperature for 30 minutes.
② Add 1ml 0. 1mol/L DTT, 2ml 1.5mol/L dNTP mixture, 0.5ml32P dATP and 2ml(2IU) T7DNA polymerase mixture into each test tube, and leave it at room temperature for 5min.
③ According to the label, add 3 ml of one of the four ddNTP termination mixtures to each test tube.
④ After a short centrifugation, keep the temperature at 37℃ for 5 minutes.
⑤ Add 5ml formamide loading buffer, heat it at 80℃ for 2min, then load it, and quickly put it on an ice bath. Take 3 ml of each sample and apply it to electrophoresis.
⑥ Read out the nucleotide sequence of the synthetic chain.
Extended data
Matters needing attention in electrophoresis and sequencing reading of sequencing reactants in extension and termination reactions;
(1) gradient gel was prepared according to the method of ordinary polyacrylamide gel.
(2) Add each sample in the order of G, A, T and C, with each lane sample of G, A and T 1ml and the lane sample of C1.5ml. ..
(3) After loading the sample, apply a pressure of 1700V for electrophoresis, and determine the electrophoresis time according to the migration of bromophenol blue and xylene blue dyes in the sample.
(4) After electrophoresis, rinse in glacial acetic acid at 65438 00℃ for 30 minutes to remove urea.
(5) After drying at 60℃ or 80℃ for 30 minutes, read the sequence by autoradiography.
Baidu Encyclopedia-dideoxy chain termination method