Molecular marker, primers and method for estimating right-end length of N introgressed segment of tobacco
A technology of molecular markers and fragments, applied in the field of molecular biology, can solve the problems of lack of technical means, limitation of N gene application, unclear length of N-introduced fragments and accompanying redundant genes, etc., to reduce burden, reduce production, and reduce the range Effect
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[0058] 1. DNA extraction
[0059] Tobacco genomic DNA was extracted respectively by conventional CTAB method, the method is as follows:
[0060] (1) Weigh about 100 mg of tobacco leaf and place it in a 1.5 mL centrifuge tube, add liquid nitrogen and grind it to powder with a pestle;
[0061] (2) Add 900 μl of 2×CTAB buffer (Tris-HCl pH 7.5100 mM, EDTA 20 mM, NaCl 1.4 M, CTAB mass percentage concentration 2%) preheated to 65°C, take it out of the water bath at 65°C for 20 minutes, and cool it;
[0062] (3) Add 200 μl of chloroform-isoamyl alcohol mixture (the volume ratio of chloroform and isoamyl alcohol is 24:1) and shake well, centrifuge at 4°C for 10 min (7200 rpm) and transfer the supernatant to a 1.5 mL EP tube;
[0063] (4) Add 200 μl of chloroform-isoamyl alcohol mixture again (the volume ratio of chloroform and isoamyl alcohol is 24:1), shake well, and centrifuge at 4°C for 10 min (7200 rpm);
[0064] (5) Take out the supernatant and place it in a new EP tube, add 1 / ...
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