Method for constructing wheat SSR (single sequence repeat) fingerprint
A fingerprint and construction method technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as heavy workload, and achieve the effects of convenient operation, less environmental impact, and improved test efficiency.
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Embodiment 1
[0021] 1) DNA extraction
[0022] Table 192 Wheat Varieties
[0023]
[0024]
[0025]
[0026] The DNA of the sample of the tested variety was extracted by CTAB method: take 0.2 g of the young tissue mixture, put it into a 1.5 mL centrifuge tube, and grind it in liquid nitrogen. Or grind the seeds and put them in a 1.5mL centrifuge tube. Preheat the DNA extraction solution to 65°C, add 400 μL to each tube, and mix the sample evenly. Place the centrifuge tube in a metal bath or water bath at 65°C, keep it warm for 30 minutes, and then remove it. Add 400 μL of 24:1 chloroform-isoamyl alcohol (V / V) to the centrifuge tube and shake to mix. Centrifuge at 10000g for 10min. Transfer 200 μL of the supernatant to another 1.5 mL centrifuge tube, add 400 μL of -20°C pre-cooled absolute ethanol to precipitate the DNA. Centrifuge at 10,000 g for 1 min, discard the supernatant, add 500 μL ethanol-ammonium acetate solution, and centrifuge at 6,000 g for 5 min to collect the pr...
Embodiment 2
[0044] Using 25 pairs of core primers (see Table 2, Table 3) to detect 92 wheat varieties (see Table 1) on denatured polyacrylamide gel. The specific operation steps of the test are as follows
[0045] 1) DNA extraction: Same as Example 1.
[0046] 2) PCR amplification: same as Example 1.
[0047] 3) Denaturing PAGE electrophoresis
[0048] a. Pre-electrophoresis
[0049] Install the gel plate on the electrophoresis tank, add about 800mL of 0.25×TBE buffer preheated to 65°C to the upper tank to make it about 3cm above the top of the gel, and add 800mL of 1×TBE buffer to the lower tank. Under 60W constant power, pre-electrophoresis for 10-20min.
[0050] b. Sample preparation
[0051] Add 10 μL of loading buffer to 25 μL of the PCR sample and mix well. Denature in a water bath or a PCR instrument at 95°C for 5 minutes, take it out, and quickly place it on crushed ice.
[0052] c. Electrophoresis
[0053] Rinse the top of the gel several times with the buffer drawn into ...
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