High-flux plant tissue genome DNA extraction method based on paramagnetic particle method
A plant tissue and extract technology, applied in the field of molecular biology, can solve the problems of low scale standardization, many manual operation steps, pollution of the environment, etc., and achieve the effects of stable and reliable DNA quality, reduced quality impact, and high safety.
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Embodiment 1
[0036] Example 1 Establishment of a high-throughput genomic DNA extraction method for plants based on the magnetic bead method
[0037] (1) Take about 0.10 g of the plant leaves to be extracted (one seed is one), put them in a 2.00 mL 96 deep-well plate and put steel balls with a diameter of 0.4 cm. The 96-deep well plate was placed in a -80°C ultra-low temperature freezer for one hour.
[0038] (2) Place the 96-well plate in a high-throughput grinder and grind at a frequency of 1500 rpm for 30s (the seeds are ground twice using this condition).
[0039] (3) Add 400 μL of 2×CTAB extract to the deep-well plate, mix well, and bathe in water at 65°C for 30 minutes; the formula of the 2×CTAB extract is: 1.40mol / L NaCl, 0.10mol / L Tris-HCl, 0.02 mol / L EDTA, 20.00g CTAB, 5.00g sorbitol, 10.00g PVP. (Seeds use SDS extract, the SDS extract formula is: 0.70mol / L NaCl, 0.05mol / L Tris-HCl, 0.01mol / L EDTA, 10.00g SDS, 5.00g sorbitol, 10.00g PVP).
[0040] (4) Put the sample after the wa...
Embodiment 2
[0073] About 0.10 g of cotton leaves to be extracted was placed in a 2 mL 96 deep-well plate and steel balls with a diameter of 0.40 cm were placed. The 96 deep-well plate was frozen in a -80°C ultra-low temperature refrigerator for one hour, then taken out, and ground in a high-throughput grinder with a frequency of 1500r for 30s.
[0074] Add 400 μL of 2×CTAB extract solution to the deep-well plate, mix well, and heat in a water bath at 65°C for 30 min; put the sample after the water bath into a centrifuge, and centrifuge at 4500 rpm for 1 min. Add 200 μL of deproteinized solution to the deep-well plate, set the oscillator frequency to 1000 rpm for 10 seconds, then centrifuge at 4500 rpm for 10 min; put the deep-well plate into the centrifuge and centrifuge at 4500 rpm for 1 min. Place the centrifuged deep-well plate in the liquid transfer workstation to open the nucleic acid extraction program, and place the magnetic frame, absolute ethanol, 70% ethanol, magnetic beads, ddH...
Embodiment 3
[0082] Take one cotton seed to be extracted, place it in a 2mL 96 deep-well plate and put a steel ball with a diameter of 0.40cm. The 96 deep-well plate was frozen in a -80°C ultra-low temperature refrigerator for one hour, then taken out, and ground in a high-throughput grinder with a frequency of 1500r for 1 min.
[0083] Add 400 μL of SDS extract solution to the deep-well plate, mix well, and heat in a water bath at 65°C for 30 min; put the sample after the water bath into a centrifuge, and centrifuge at 4500 rpm for 1 min. Add 200 μL of deproteinized solution to the deep-well plate, set the oscillator frequency to 1000 rpm for 10 seconds, then centrifuge at 4500 rpm for 10 min; put the deep-well plate into the centrifuge and centrifuge at 4500 rpm for 1 min. Place the centrifuged deep-well plate in the liquid transfer workstation to open the nucleic acid extraction program, and place the magnetic frame, absolute ethanol, 70% ethanol, magnetic beads, ddH in sequence accordi...
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