Molecular marker, primers and method for estimating left-end length of N introgressed segment of tobacco
A technology of molecular markers and fragments, which is applied in the field of molecular biology, can solve the problems of restricting the application of N genes, lack of technical means, and lack of breakthrough progress in breeding resistant TMV tobacco, and achieve the effect of reducing yield reduction and reducing burden
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[0063] 1. DNA extraction
[0064] To extract tobacco genomic DNA by conventional CTAB method, the method is as follows:
[0065] (1) Weigh about 100 mg of tobacco leaves and place them in a 1.0 mL centrifuge tube, add liquid nitrogen and grind them with a pestle to powder;
[0066] (2) Add 900μl of 2×CTAB buffer (Tris-HCl pH 7.5100mM, EDTA20mM, NaCl1.4M, CTAB mass percentage 2%) preheated to 65°C, take out and cool in a 65°C water bath for 20 minutes;
[0067] (3) Add 200μl of chloroform-isoamyl alcohol mixture (the volume ratio of chloroform and isoamyl alcohol is 24:1) and shake well, centrifuge at 4°C for 10min (7200rpm) and transfer the supernatant to a 1.0mL EP tube;
[0068] (4) Add 200 μl of chloroform-isoamyl alcohol mixture (the volume ratio of chloroform and isoamyl alcohol is 24:1) again, shake it, and centrifuge at 4°C for 10 min (7200 rpm);
[0069] (5) Take out the supernatant and place it in a new EP tube, add 1 / 10 of the supernatant volume concentration of 3M pH5.2 sodium...
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