Extraction and identification of proton DNA.

the extraction and identificationexperimental purposeof the three- particles DNA in this experimentthrough this experiment, learning and mastering the extraction of prosultes by alkali lysing method.. The -alkalilysateextraction of prosultes is based on the topological differences between co-priced closed ring-like proton DNA and linear chromosomal DNA. In the narrow range of pH between 12.0 and 12.5, linear DNA double helix structure is unzipped and denatured, although under such conditions, the hydrogen bonds of co-priced closed-loop granulated DNA are broken, but the two complementary chains are coiled around each other and still bind tightly together. When the potassium acetate high salt buffer added to pH4.8 is restored to Ph to neutral, the two complementary strands of co-priced closed ring-like granule DNA remain together, so the complexity is fast and accurate, and the two complementary chains of the linear chromosome DNA They are completely separated from each other, and the complexity is less rapid and accurate, and they wrap around to form a mesh structure that is removed by centrifugation, chromosomal DNA and unstable large molecule RNA,
protein
-SDS complexes, etc.. Theof experimental instruments and equipment1.1.
thermostat
culture
box 2.thermostat shaker 3. table centrifuge (maximum speed 4000rpm) 4.frozen high-speed centrifuge 5.autocultural sterilization pot 6.ultra-clean workbench 7.micro
leep
8.eppendorf tupe, tip
8.eppendorf tupe, tip) 1. Glucose 2.trihydroxymethymeethane (Tris)3. EDTA) 4.sodium hydroxide 5.12ane sodium sulfate (SDS) 6.potassium oxide 7.ice beta acid 8.chloroform.. 9.Ethanol 10.pancreatase 11.ampicillin 12.sucrose 13.brominol blue 14.phenol 15.β base ethanol 16. Hydrochloric acid 17. E. colicontaining pUC18 reagents
preparation 1. Solution I.
50mmol/L glucose 5mmol/L trihydroxymethane (Tris) Tris. HCl (pH8.0) 10mmol/L ethyl diamine tetacetic acid (EDTA) (pH8.0) 2. Solution II. 0.4 mol/L NaOH, 2% SDS, Mixing 3.solution III. 5mmol/L potassium acetate 60 ml ice acetic acid 11.5 ml water 28.5 ml 4.TE buffer 10mmol/L Tris HCl 1 mmol/L EDTA (pH8.0) 5.70% ethanol (in a refrigerator at -20 degrees C, put back when used) 6.Pancreatic RNA enzyme dissolves RNA enzymes in 10mmol/L Tris. In HCl (pH7.5), 15mmol/L NaCl, with a concentration of 10 mg/ml, heat 15min at 100C, cool slowly to room temperature, and store at -20C.

. 7. Termination fluid: 40% sucrose, 0.25% bromo blue phenol 8.phenol experimental steps ) (i) extracted prosuldiates 1. Add 2 ml of LB liquid
f medium
containing the appropriate antibiotics to the test tube, connect to E. coli containing the granules, and oscillate culture overnight at 37 degrees C.
. 2. Take 1.5 ml of culture and pour it into a trace centrifuge tube, 4000rpm, centrifuge 2min.
. 3. Absorb the culture fluid so that the cells precipitate as
as
. . 4. The bacterial precipitation is suspended in a solution of 100 sl I. and mixed well, with 10 min at room temperature. . 5. Add 200 μl solution II. (fresh preparation), mix the contents and place the centrifuge tube on ice for 5 min.
. 6. Add 150 μl solution III. (pre-cooling on ice), cover the tight tube port, upside down several times to mix well. . 7. 1200rpm, centrifuged 15 min, transferred to another centrifuge tube. . 8. Add iso-volume phenols to the upper clear: chloroform (deprotein), mix repeatedly, 12000rpm, centrifugal 5min, transfer the upper clear to another centrifugal tube. . 9. Add 2x volume ethanol to the top clear, mix well, and place 5-10min at room temperature. 12000rpm Centrifuge 5min. Pour over the liquid, pour the centrifugal tube upside down on the absorbent paper and absorb the liquid. . 10. Wash the granule DNA precipitation with 1 ml70% ethanol, oscillate and centrifuge, pour over the liquid, vacuum dry or dry in the air. . 11. Add a 50 sl TE buffer, which contains 20 sg/ml of pancreatic RNA enzyme, so that the DNA is completely dissolved and preserved at -20 degrees C.
. (ii)
Agar
sugar gel electrophoresis detection DNA method see experiment II . What is the principle of extracting proton DNA by alkali lysis? . 2. Take a photo of the prosurage DNA electrophoresis map with the gel imaging system and analyze the stripe of electrophoresis a total of 12 school hours, students (6 hours) Afternoon 1st - 2nd morning: with LB culture, inoculation, liquid culture 2nd afternoon: extraction of prosalytic DNA Night 2: Testing of .