Rapid small volume extraction of total plant DNA
Rapid small volume extraction of total plant DNA
DNA molecule is the basic material of molecular biology research, according to different experimental purposes can be taken to different extraction methods to obtain the quantity and quality of DNA. the purpose of the experiment is to understand the main methods of plant DNA extraction, master the CTAB method of rapid extraction of rice DNA.
Operation method
CTAB method
Principle
CTAB method: This method is simple, fast and has a high DNA yield (slightly less pure, suitable for general molecular biology operations). CTAB is a non-ionic decontaminant. plant material is treated with CTAB in combination with a water bath at 65 °C so that cells are lysed, proteins are denatured, and DNA is released. cTAB forms a complex with nucleic acids, which is soluble and stable at high salt (>0.7 mM) concentrations, but at low salt concentrations (0.1-0.5 mM NaCl) the CTAB-nucleic acid complexes are However, at low salt concentrations (0.1-0.5 mM NaCl), the CTAB-nucleic acid complexes precipitated due to reduced solubility, while most of the proteins and polysaccharides remained dissolved in the solution. After centrifugation and discarding the supernatant, the CTAB-nucleic acid complex is soaked in 70-75% alcohol to remove the CTAB, and then extracted with chloroform/isoamyl alcohol (24:1) to remove proteins, polysaccharides, and pigments to purify the DNA, and then finally precipitated from the DNA by DNA precipitants such as isopropanol or ethanol.
Materials and Instruments
Rice Leaf Move 1. Collect appropriate amount of young leaves, powdered with liquid N2, 0.4 g in 1.5 ml centrifuge tube (-20 ℃ pre-cooled). Caveat 1. take as young leaves as possible, if too old and phenolic, must be treated with 10 mM β-ME. 2. pre-freeze the mortar and do not melt the powder until CTAB is added. 3. 24:1 chloroform/isoamyl alcohol extraction should be done gently, and the tip of the transfer lance should preferably be cut wide. 4. All reagents used must be sterilized, gloves. For more product details, please visit Aladdin Scientific website.
Liquid nitrogen CTAB Chloroform Isoamyl alcohol Ethanol NH4AC EtOH
Centrifuge Tube Centrifuge
2. Preheat 1.5×CTAB to 95 ℃, add 1 ml to the centrifuge tube containing leaf powder and mix well (prevent freezing and thawing).
3. Immediately place in a water bath at 65 °C for 30 min, every 5 min, upside down.
4. Centrifuge at 12000 g for 5 min.
5. Aspirate about 600 μl of the supernatant and add an equal volume (600 μl of supernatant ) of chloroform/isoamyl alcohol (24:1), up and down several times until the lower liquid phase is dark green.
6. Centrifuge at 12000 g for 5 min.
7. Take 450 7. Take 450 μl of the supernatant into a new 1.5 ml tube. supernatant in a new 1.5 ml centrifuge tube, add 1 ml of 95% ethanol and 45 μl (10 M NH4AC ), mix well and leave at room temperature for 10 min.
8. Centrifuge at 12000 g for 10 min, remove the supernatant, wash the precipitate with 75% EtOH and dry naturally for about 30 min.
9. Add 50 μl 1/10 TE or sterile water (containing 20 mM/RNase), place at 4 ℃ overnight, and wait until the DNA is solubilized, then test the DNA concentration and quality.