High-throughput extraction method for sugarcane leaf genome by using ball mill

A sugarcane leaf, ball mill technology, applied in recombinant DNA technology, DNA preparation and other directions, can solve the problems of low purity, troublesome sample grinding, low extraction amount, etc., and achieve the effect of simplifying experiments, less sampling amount, and wide application range.

Inactive Publication Date: 2010-12-01
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention proposes a high-throughput extraction method of sugarcane leaf genome using a ball mill, and optimizes the separation technology for the particularity of sugarcane, thereby overcoming the disadvantages of troublesome sample grinding, low extraction volume, and low purity in the preparation of sugarcane DNA templates.

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  • High-throughput extraction method for sugarcane leaf genome by using ball mill
  • High-throughput extraction method for sugarcane leaf genome by using ball mill

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Embodiment 1

[0020] The specific implementation steps of high-throughput extraction of sugarcane leaf genome by using a ball mill are (1) according to the number of samples, put one 4 mm diameter stainless steel bead into each of 20 2 mL centrifuge tubes; (2) take 50 mg of fresh sugarcane Put the leaves into each centrifuge tube, cover tightly and pre-freeze in liquid nitrogen for 30 min; (3) After taking it out, quickly put it on a 45-hole grinding stand with a pre-frozen ball mill at -20°C, grind it in the ball mill for 30 s, and take it out Place the centrifuge tubes on ice; (4) add 1.0 mL of 2% CTAB extraction buffer preheated at 65°C to each centrifuge tube; (5) bathe in water at 65°C for 40 min, and centrifuge by inverting and mixing every 5 min (6) Add 800 μL of phenol: chloroform: isoamyl alcohol mixture (volume ratio 25:24:1) to each tube, invert and mix until milky white, let stand at room temperature for 10 min, centrifuge at 12,000 r / min for 10 min, and take 800 μL Transfer the s...

Embodiment 2

[0022] (1) According to the number of samples, put one 4 mm diameter stainless steel bead into each of 35 2 mL centrifuge tubes; (2) Take 75 mg of fresh sugarcane leaves and put them into each centrifuge tube, tightly cap and place in liquid nitrogen (3) After taking it out, quickly put it on the 45-hole grinding stand of a pre-frozen ball mill at -20°C, grind it in the ball mill for about 35 seconds, take out the centrifuge tube and put it on ice; (4) Put each centrifuge tube Add 1.0 mL of 2% CTAB extraction buffer preheated at 65°C; (5) 65°C water bath for 40 min, and mix the centrifuge tube by inversion every 5 min; (6) Add 800 μL of phenol:chloroform:iso Amyl alcohol mixture (volume ratio 25:24:1), invert and mix until milky white, let stand at room temperature for 10 min, centrifuge at 12,000 r / min for 10 min, transfer 800 μL supernatant to a new centrifuge tube (repeatable pumping) (7) Add 2 / 3 volume of isopropanol pre-cooled at -20°C, mix upside down, centrifuge at 12,0...

Embodiment 3

[0024] (1) Put one 4 mm diameter stainless steel bead into each of 45 2 mL centrifuge tubes according to the sample quantity; (2) Put 100 mg of fresh sugarcane leaves into each centrifuge tube, cap tightly and place in liquid nitrogen (3) After taking it out, quickly put it on the 45-hole grinding stand of a pre-frozen ball mill at -20°C, grind it in the ball mill for about 40 s, take out the centrifuge tube and put it on ice; (4) Centrifuge each Add 1.0 mL of 2% CTAB extraction buffer preheated at 65°C to the tube; (5) bathe in water at 65°C for 40 min, and invert the centrifuge tube every 5 min during this period; (6) add 800 μL of phenol:chloroform to each tube: Isoamyl alcohol mixture (volume ratio 25:24:1), invert and mix until milky white, let stand at room temperature for 10 minutes, centrifuge at 12,000 r / min for 10 minutes, transfer 800 μL supernatant to a new centrifuge tube (repeatable (7) Place the centrifuge tube in a -20°C freezer for 1 h, use a clean tip or a to...

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Abstract

The invention discloses a high-throughput extraction method for sugarcane leaf genome by using a ball mill, and relates to technology for separating genomic DNA of leaves of sugarcane by using the ball mill aiming at the particularity of the sugarcane. Based on the characteristic that the ball mill changes a magnetic field to grind a plurality of sugarcane leaf samples by metal balls at one time, parameters such as diameter of steel balls, liquid nitrogen pre-processing time, using amount of leaves, grinding time and the like are optimized, and a method applicable to mass extraction of the sugarcane leaf genome is established. The method mainly comprises the steps of sampling, pre-freezing, grinding, chemical extraction and the like. The method overcomes the disadvantages of time-consuming and labor-consuming sample grinding, complex operation, high using amount of leaves, high cross contamination rate, low efficiency and the like existing in the conventional preparation of the sugarcane genomic DNA, has the advantages of wide application range, simplicity, convenience, high speed, low contamination rate, small sample number and the like and is particularly suitable for rapidly preparing the sugarcane leaf genome in batches.

Description

technical field [0001] The invention belongs to the field of molecular biology, and more specifically relates to a method for high-throughput extraction of sugarcane leaf genome by using a ball mill. Background technique [0002] The extraction of plant DNA is different from that of animals. Plant cells and tissues have hard cell walls and often contain a large amount of impurities such as RNA, protein, polysaccharides, tannins and pigments. These problems bring many difficulties to the extraction and purification of DNA. In practice, most proteins can be denatured and removed by precipitation after treatment with phenol, chloroform, etc., and most RNA can be removed by RNase A. However, polysaccharide impurities are generally difficult to remove. When the concentration of these impurities is high, the DNA extract is often gelatinous, and at low concentrations, it will also interfere with subsequent operations and quantitative analysis of nucleic acids by spectrophotometry. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 陈平华方静平许莉萍王恒波陈由强陈如凯
Owner FUJIAN AGRI & FORESTRY UNIV
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