Improved CTAB method for extracting nucleic acid of paulownia fordii
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A technology of tung nucleic acid and lotus leaf, applied in the field of molecular biology, can solve the problems of pipe tip drawing, incomplete cell lysis, unclear main DNA band, etc., and achieve the effect of reducing loss.
Pending Publication Date: 2021-11-02
HAINAN NORMAL UNIVERSITY
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Problems solved by technology
[0010] The technical problem to be solved by the present invention is: how to provide a kind of method that is suitable for extracting the nucleic acid of the chrysanthemum chrysanthemum, solve the incomplete cell lysis that occurs when
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Embodiment 1
[0029] (1) an appropriate amount of fresh leaves lotus Tong, has been placed in pre-chilled mortar and good, while adding liquid nitrogen grinding edge, the blade after grinding powder 200mg taken sufficiently transferred to the preheated to 65 deg.] C 800μL CTAB lysis buffer ( 2% (m / v) CTAB, 0.1mol / LTris-HCl, 20mmol / L EDTA, 1.4mol / LNaCl, 2% (v / v) β-mercaptoethanol), the tubes are placed in the shock vortex 1min, after mixing into the water bath, the bath temperature set 65 ℃, water bath 40min, 10min a reversed spacer shake.
[0030] After cooling into room (2) heating is completed, and then added to an equal volume of chloroform-isoamyl alcohol = 24︰1 ︰ sufficiently mix by inversion, centrifuged at a speed of 10 ~ 15min 11000 ~ 13500rpm at room temperature;
[0031] (3) the supernatant was added 2 / 10 volumes of 3mol / L of a solution of NaAc and 2 volumes of 95% ethanol, mix by inversion slight precipitation after 40 ~ 60min -20 deg.] C, at a speed of 11000 ~ 13500rpm ,...
Embodiment 2
[0034] (1) an appropriate amount of fresh lotus for Leaves placed prechilled mortar, thoroughly ground in liquid nitrogen was added. The powder was transferred to a centrifuge tube 2mL added preheating temperature is 65 deg.] C the lysate, mixing, addition of RNA enzyme, vortexing 1min, 65 deg.] C water bath after mixing water bath 40min, every 10min shaking upside uniform again. Lysates composition: 2% (m / v) CTAB, 0.1mol / LTris-HClpH8.0,20mmol / L EDTA, 1.4mol / L NaCl, 2% (m / v) β-mercaptoethanol, 2% (m / v) PVP;
[0035] (2) allowed to cool to room temperature, an equal volume of phenol Tris balance ︰ ︰ chloroform isoamyl alcohol = 25︰24︰1, centrifuged at a speed of 11,000 to 10 ~ 15min at 13500rpm at room temperature;
[0036] (3) The supernatant was transferred to a new tube, 1 / 10 volumes of 3mol LNaAc equal volume of isopropanol and / , mix placed at -20 ℃ 40 ~ 60min, at a speed of 11000 ~ 13500rpm4 ℃ centrifugation 10 ~ 15min;
[0037] (4) The supernatant was discarded, ...
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Abstract
The invention belongs to the field of molecular ecology experiments, and particularly provides a CTAB (cetyltrimethyl ammonium bromide) extraction method for an improved genome DNA (deoxyribonucleic acid) version of an endangered semi-mangrove plant tung paulownia. The endangered semi-mangrove plant paulownia fortunei is rich in polysaccharide and polyphenol, so that the tissue grinding fluid is extremely viscous, and the extraction of nucleic acid with good quality is extremely difficult. On the basis of a traditional CTAB method, cells are split by adopting an improved lysis solution, and extraction is carried out through a mixed solution of Tris balanced phenol-chloroform-isoamyl alcohol in a certain proportion. The DNA is precipitated by using sodium acetate-isopropanol with improved volume, and then the DNA is purified so as to obtain the high-quality genome DNA. The DNA extracted by adopting the method for extracting the nucleic acid of the endangered semi-mangrove plant tung paulownia, provided by the invention, is high in concentration and purity and is not easy to degrade, and most importantly, polysaccharide and polyphenol can be effectively removed. A foundation is laid for subsequent molecular biology experiments such as PCR amplification, molecular marker technology development, genome library construction and the like.
Description
Technical field [0001] The present invention is in the field of molecular biology, particularly relates to an endangered mangrove lotus Tong modified version CTAB method to extract nucleic acid. Background technique [0002] Lotus Tong (Hernandia nymphaeifolia (J.Presl) Kubitzki) is hernandiaceae, hernandia evergreen trees, lotus Tong material because of its rich polysaccharides, polyphenols, high protein, special ingredients, extracts extremely viscous and easy browning, so nucleic acid extraction more difficult. [0003] The traditional CTAB method as follows: [0004] (1) Weigh 0.2g samples were pulverized in liquid nitrogen and placed in 2ml centrifuge tube, was added a CTAB solution (2% (m / v) CTAB, 0.1mol / LTris-HClpH8 800uL preheated by 65 ℃. 0,20mmol / LEDTA, 1.4mol / LNaCl). after thorough mixing, which was blended in a heated water bath 65 deg.] C 40min, 10min each shake reversed several times. [0005] (2) cooling to room temperature was added an equal volume of chlo...
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