Experimental principle Experimental reagent Reagent preparation Laboratory equipment 7. Surgical scissors, forceps, absorbent paper Experimental material animal tissue block Experimental procedure Rose Tea, Dried Rose, Dried Rose Buds, Rose Flower Tea, Air Dried Rose Jiangsu Tiankang Food Co., Ltd. , https://www.tiankangfood.com
DNA is an important component of all biological cells and is mainly found in the nucleus. The cells are disrupted by grinding and SDS; phenol and chloroform can denature the protein, and the mixture is repeatedly extracted (phenol: chloroform: isoamyl alcohol) to denature the protein, and then centrifuged to remove the denatured protein; RNase degrades the RNA to obtain pure DNA molecule.
1. Saline
2. Sodium dodecyl sulfate (SDS)
3. Tris
4. Ethylenediaminetetraacetic acid (EDTA)
5. Saturated phenol
6. Chloroform
7. Isoamyl alcohol
8. Absolute ethanol
9.75% ethanol
10. Proteinase K
11. RNase enzyme
1. Tris–HCL 1mol/L PH8.0 50ml
Preparation method:
40ml double distilled water, 6.057g solid Tris dissolved in a beaker, adjust the pH value to 8.0 with concentrated hydrochloric acid, transfer to a 50ml volumetric flask, add double distilled water to volume, shake well, transfer to the prepared infusion bottle Labeled, autoclaved, cooled to room temperature, and stored at 4 ° C for later use.
2. Saline: 0.85% NaCL 100ml
Preparation method:
Dissolve 0.85g of solid NaCL in 20ml double distilled water, dilute to 100ml with water, shake well, transfer to the prepared infusion bottle, label it, autoclave, drop to room temperature, and store at 4 °C for use.
3. EDTA 0.5mol/L PH8.0 50ml
Preparation method:
Dissolve 9.08g of EDTA·Na2·2H2O in 40ml of double distilled water, adjust the pH to 8.0 with 1g of NaOH granules (add slowly), and make up to volume with 50ml volumetric flask. If EDTA is insoluble, first add NaOH to dissolve. Then gradually add EDTA·Na2·2H2O.
4. TES buffer (release DNA) 100ml
Preparation method:
Dissolve 0.5844 g of 5 mol/l NaCl in 80 ml of double distilled water, add 1 ml of 0.5 mol/l EDTA, 0.2 ml of Tris-HCl (pH=8.0), add a volume to 100 ml, shake well, and transfer to The prepared infusion bottle is labeled, and after autoclaving, it is cooled to room temperature and stored at 4 ° C for use.
5.10% SDS (denaturing agent breaking cell wall) 100ml
Preparation method:
Dissolve 10 g of sodium dodecyl sulfate (SDS) in 80 ml of double distilled water and dissolve at 68 ° C, adjust to pH = 7.2 with concentrated HCl, dilute to 100 ml, shake well, and transfer to the prepared infusion bottle. , label it, save it at 4 °C.
6. Protease K (degraded protein): 20 mg/mL dissolved in sterile trihydrate.
7. RNase (degrading RNA)
Preparation method:
The pancreatic RNase (RNase A) was dissolved in 10 mmol/L Tris·CL (pH 7.5) and 15 mmol/L NaCL to prepare a concentration of 10 mg/ml, heated at 100 ° C for 15 min, slowly cooled to room temperature, and dispensed. Small portions are stored at –20 °C.
8. Chloroform: isoamyl alcohol = 24:1 100ml
Add chloroform and isoamyl alcohol in a ratio of 24:1, shake well, transfer to the prepared bottle, label it, and store at 4 °C for later use.
9. TE buffer (dissolved DNA) PH8.0 50ml
Preparation method:
Add 0.5 ml of 10 mmol Tris-HCl (pH 8.0), 0.1 ml of 0.5 mol/l EDTA (pH 8.0) to a 50 ml volumetric flask, adjust the pH to a volume of 50 ml, shake well, and transfer to preparation. In a good bottle, label it, after autoclaving, drop to room temperature and store at 4 °C for later use.
1. High speed centrifuge
2. Oven
3. refrigerator
4. Water bath
5. Micropipette
6. Autoclave
8. Micro-pump
9. Rinse, 1.5mL centrifuge tube, disposable gloves, 1.5mL centrifuge tube holder, marker pen, etc.
1. The tissue block was thawed, the blood stain was washed away with physiological saline, about 0.5 g of tissue was cut out, placed in a 1.5 ml centrifuge tube, and cut.
2. Add 0.45ml TES to mix, add 50ul SDS (10%), 5.0ul proteinase K (20mg/ml), mix well, heat at 56 ° C for 4-6h, shake once every 2h.
3. After standing at room temperature, add an equal volume of saturated phenol (500 ul), mix by inversion, 10000 r/m, centrifuge for 10 m, separate the aqueous phase and the organic phase, carefully pipet the upper aqueous phase containing the nucleic acid into a new 1.5 ml centrifuge tube.
4. An equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was added, mixed by inversion, 10000 r/m, centrifuged for 10 minutes, and the upper layer was transferred to a new 1.5 ml centrifuge tube.
5. Add an equal volume of chloroform: isoamyl alcohol (24:1), mix by inversion, 10000r/m, centrifuge for 10 minutes, and take the supernatant to a new 1.5ml centrifuge tube.
6. The DNA was precipitated by adding 2.5 volumes of pre-cooled absolute ethanol at -20 ° C to observe the phenomenon.
7.12000 r/m, centrifuge for 10 minutes, discard ethanol.
8. Wash with 75% ethanol stored at -20 ° C, 10000 r / m, centrifuge for 5 minutes, remove ethanol, and dry the DNA at 55 ° C.
9. Add appropriate amount of TE to dissolve DNA (depending on the amount of DNA), and store at -20 °C for later use.
Precautions
1. The force of each step should be softened to prevent damage to DNA by mechanical shearing force.
2. When taking the supernatant, be careful not to pick up the middle protein layer.
3. Do not sway DNA when rinsing with ethanol to remove ethanol.
4. After centrifugation, do not shake the centrifuge tube, keep the tube steady, and the slope is facing outward.