Plant DNA extraction and quantitative analysis experiments
Plant DNA extraction and quantitative analysis experiments
The cells of young tissues are in the stage of exuberant division, with larger nuclei and less cytoplasm, high concentration of nucleic acid, and fewer inclusions, fewer secondary metabolites, and relatively fewer proteins and polysaccharides. In the presence of SDS or CTAB substances, the mechanical milling, which ruptures the cells and releases the inclusions, the extracted DNA and RNA have a high yield and good purity.
Operation method
Plant DNA extraction and quantitative analysis experiments
Principle
The cells of young tissues are in the stage of exuberant division, the nucleus is larger and the cytoplasm is less, the concentration of nucleic acid is high and there are fewer inclusions, fewer secondary metabolites, and relatively fewer proteins and polysaccharides.In the presence of SDS or CTAB substances, the cells are ruptured by mechanical milling and the inclusions are released, and the extracted DNA and RNA have high yields and are of good purity.The extracting solution used for DNA quantification can be analyzed by UV spectrometry. The extraction solution, pipette tips, centrifuge tubes, etc. used for DNA extraction need to be autoclaved to inactivate Dnase. quantitative analysis of DNA can be done by UV spectroscopy. The principle is that DNA molecules have specific UV absorption peaks at 260 nm, and the intensity of absorption is proportional to the concentration of DNA. In addition, it can be analyzed by the brightness of the DNA bands shown on agarose gel electrophoresis, because EB, as a fluorescent dye, can insert between the base pair planes of DNA and bind to it, producing fluorescence under the excitation of UV light, and the amount of EB on a DNA molecule is directly proportional to the length and number of DNA molecules. The amount of EB on a DNA molecule is directly proportional to the length and number of DNA molecules. When a known concentration of DNA Marker is added during electrophoresis as a reference for the relative molecular mass and concentration of DNA, the fluorescence intensity of the sample DNA can roughly indicate the amount of DNA. The advantage of this method is that it is simple and easy to perform, and can be combined with agarose gel electrophoresis to analyze the integrity of the DNA sample; the disadvantage is that it is not very accurate.
Materials and Instruments
Young Plant Tissue Move I. Experimental materials and supplies Caveat Gentle handling of DNA solutions and rapid freezing of plant tissues are important to mitigate mechanical shear and nucleic acid digestion without oscillation.EB is a strong mutagen, and phenol, chloroform, and CTAB are corrosive or toxic. Gloves must be worn when using solutions containing these drugs, and centrifuge tubes containing phenol and chloroform should be disposed of centrally or discarded. For more product details, please visit Aladdin Scientific website.
CTAB extract
Centrifuge Centrifuge tubes UV spectrophotometer Micropipettes Pipette tips Pipette paper Water bath Refrigerator Mantle Liquid nitrogen tank Electronic balance pH meter Autoclave Disposable gloves and masks Electrophoresis apparatus and electrophoresis tanks
Young plant tissues (e.g., young leaves, flower organs, young roots, etc.).
Centrifuge, centrifuge tube, UV spectrophotometer, micropipette, pipette tips, blotting paper, water bath, refrigerator, mortar and pestle, liquid nitrogen tank, electronic balance, pH meter, autoclave, disposable gloves and masks, electrophoresis apparatus and electrophoresis tank.
CTAB extraction solution: 2% CTAB (m/V), 100 mmol/L Tris-HCl pH 8.0, 20 mmol/L EDTA pH 8.0, 1.4 mol/L NaCl, 2% PVP (add 2% PVP after sterilization to make it fully dissolved). 1% (V/V) β-mercaptoethanol was added before grinding the young plant tissues.
TE (pH 8.0): weigh 1.211 g Tris, 0.372 g EDTA-Na2, dissolve with 800 mL distilled water by heating and stirring, adjust the pH 8.0 with hydrochloric acid, and then volume to 1000 mL with distilled water. autoclave for 20 min.
Chloroform/isoamyl alcohol (24:1, V/V): Mix chloroform and isoamyl alcohol in the ratio of 24:1 by volume, put in a brown bottle and store at 4℃.
Phenol/Chloroform/Isoamyl Alcohol (25:24:1): Mix saturated phenol, chloroform and isoamyl alcohol in the ratio of 25:24:1 by volume, put in a brown bottle and store at 4℃.
10 mg/L of EB storage solution: add 1g of EB into 100 mL of distilled water, stir on a magnetic stirrer for a few hours to dissolve completely, transfer to a brown bottle and store at 4℃ away from light.EB is a strong mutagen, wear gloves and a mask when weighing. Once contacted with EB, rinse immediately with plenty of water.
1X TAE electrophoresis buffer: 50X TAE concentrated storage solution contains 242 g of Tris base, 57.1 mL of glacial acetic acid, 100 mL of 0.5 mol/L EDTA of pH8.0, add 600 mL of distilled water, stir on a magnetic stirrer, and finally add distilled water to finalize the volume to 1000 mL.
6X gel spiking buffer: 0.25% bromophenol blue, 0.25% xylene cyan FF, 40% (m/V) sucrose aqueous solution, stored at 4℃.
3M NaAc (pH 5.2)
II. Experimental content and experimental steps
1. Extraction of DNA
(1) The extracting solution, pipette tips, centrifuge tubes, etc. used for DNA extraction should be autoclaved in an autoclave at 121℃ (about 1.1 kg/cm2) for 20 min.
(2) Grind 50mg of young leaves into fine powder with liquid nitrogen, place it in a 1.5ml centrifuge tube and add 600μl of CTAB extract preheated to 65℃, shake gently and mix well.
(3) Add 600 μl of CTAB extraction solution into the centrifuge tube at 65℃ for 1 h. Shake gently to mix.
(4) Centrifuge at 12000 rpm for 10 min and transfer the supernatant to another 1.5 ml tube.
(5) Add an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) to the supernatant, mix gently for 10 min, then centrifuge at 12000 rpm for 10 min, then transfer the supernatant into a new tube.
(6) Add an equal volume of chloroform/isoamyl alcohol (24:1) to the supernatant, mix gently for 10 min, then centrifuge at 12000 rpm for 10 min, then transfer the supernatant into a new tube.
(7) Add 1/10 volume of 3M NaAC (pH5.2), equal volume of cold isopropanol to the supernatant and mix carefully. centrifuge at 12000rpm for 10min and discard the supernatant.
(8) Wash the precipitate once with 70% ethanol, centrifuge at 12000rpm and discard the supernatant.
(10) Blow-dry the precipitate on an ultra-clean bench and add 50 μl TE (pH 8.0) to dissolve at room temperature.
The concentration and quality of DNA were detected by electrophoresis or by UV spectrophotometer.
2. Concentration Determination
(1) Detection of DNA by agarose electrophoresis
Estimate the volume of agarose gel according to the number of sampling tubes and the size of the electrophoresis tank, weigh the agarose dry powder and prepare 1% agarose gel with 1xTAE buffer, heat the agarose on the electric stove to make the agarose fully dissolved, wait until the temperature is lowered to 60 ℃, add the EB, so that the final mass concentration of the EB is 1 μg/mL, shake well, and then poured into the bed of the gel with a comb, to avoid the production of air bubbles, about 30 minutes at room temperature, the gel solidified naturally. The gel was solidified naturally at room temperature for about 30 min.
Put the agarose gel into the electrophoresis tank and pour 1X TAE electrophoresis buffer into the tank so that the surface of the liquid is about 0.5 cm above the surface of the gel.
Insert a pipette with a 10 μl tip, inhale the mixture of DNA sample and sampling buffer, insert the tip into the bottom of the spiking well, and slowly add the DNA sample into the spiking well.
After adding the sample, connect the electrophoresis tank and the power supply correctly, with the black wire connected to the cathode and the red wire connected to the anode. The red line is connected to the anode, turn on the power supply, set the voltage and time correctly, the selection of time depends on the length of the gel and the size of the voltage. Generally, the voltage is not higher than 5 V/cm, electrophoresis for about 2 h, and stop electrophoresis when the bromophenol blue is about 1-1.5 cm away from the edge of the gel.
Remove the gel carefully, place it on a UV transilluminator, detect the presence or absence of amplified fragments and separation under UV light, and take a photo on a gel imager.
Use λDNA as the relative molecular mass size of the Marker, if the band pattern is not diffuse, and neat and bright bands appear in the corresponding position with the Marker 20kb band, it means that the genomic DNA is intact and not degraded.
(2) Determination of DNA concentration by UV spectrophotometry
Take a small amount of DNA sample to be tested and dilute 50 times (or 100 times) with TE or distilled water.
Make a blank with the dilution solution and adjust the reading of the UV spectrophotometer to zero at 260 nm, 180 nm and 310 nm.
Add the DNA sample to be tested to read the OD value at the above 3 wavelengths.
The OD260/OD280 value of pure DNA sample is about 1.8, higher than 1.8 there may be RNA contamination, lower than 1.8 there is protein contamination;
III. Experimental results and calculations 