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[Rui Sai Xiao Class] The knowledge points you should master about colorectal adenomas 
1. Separation of chromatin by polyamine method
Reagents, kits: colchicine, polyamine buffer
Experimental steps :
Synchronization of cells in mitosis
1. Incubate the cells at 37 ° C with a suitable medium containing FCS, antibiotics and other essential ingredients.
2. Add colchicine to the culture medium at a concentration of 0.06 μg/ml 10 to 16 hours before collecting the mitotic cells.
Harvest cells and lyse in polyamine buffer
3. Harvest the cells.
For suspension cultured cells (500 ml medium), the cells were harvested by centrifugation at 1500 g for 5 minutes at room temperature. The resulting precipitate was washed three times with polyamine buffer at room temperature, and the cells were collected by centrifugation at 1500 g for 5 minutes.
Polyamine buffer:
15 mmol/L Tris-HCl (pH 7.4)
0.2 mmol/L spermine
0.5 mmol/L Jingmi
2 mmol/L EDTA
80 mmoI/L KCl
0.1 mmol/L PMSF (added from 1 mmol/L stock solution prepared with 100% ethanol before use)
4. The cells were resuspended in 20 ml of ice-cold polyamine buffer containing 0.1% digitonin.
5. Use a Dounce homogenizer and a B-grinding rod to perform 10 mild homogenizations to lyse the cells (too intense homogenization can cause significant damage to the chromosomes). Alternatively, the suspension can be gently aspirated into a plastic syringe and then pushed through the 25 gauge needle (the solution should be avoided from foaming).
6. After cell lysis, remove a small amount of lysate to observe chromatin release. A drop of the lysis suspension was applied to a glass slide containing 2 μg/ml of Hoechst 33258 in isolation buffer solution for DNA staining. Chromatin was observed with a fluorescence microscope.
7. Use JS-13 (Beckman), HB-4 (Sorvall), 2150 (Herace) or a similar hanging barrel rotor to pellet the lysate at 250 g for 1 minute to remove debris.
8. Remove the supernatant containing the chromosome and centrifuge at 3000 g for 15 minutes.
9. Resuspend the chromosome-containing pellet in 5-10 ml of polyamine buffer. The prepared chromosomes can be stored at 4 °C. Morphological changes were not observed after 2 to 4 weeks of storage; however, for special purpose studies (eg, enzymatic activity, unstable proteins), the resulting chromosome should be used immediately.
Second, the separation of chromatin by aqueous phase
Reagents, kits : aqueous phase lysis buffer
Instruments, consumables : centrifuge
Experimental steps :
The medium-term repression state of suspended or monolayer cultured cells is induced as described in steps 1 and 2 of the polyamine method.
2. The cells were centrifuged at 1000 g for 10 minutes at 4 ° C and then resuspended, typically 10 ml fresh medium containing 108 cells.
3. Allow the concentrated cell suspension to cool on ice for at least 30 minutes to help complete dissociation of the mitotic complex.
4. Centrifuge the cells at 10 °C for 10 min at 10 °C.
5. The pellet was resuspended in 10 ml of 75 mmol/L KCI and incubated at 4 °C for 20 minutes for hypotonic swelling.
6. Centrifuge the cells at 4 °C 1000 r/min.
7. Resuspend the cells in 10 ml of aqueous phase lysis buffer, gently mix and place on ice for 5 minutes.
Aqueous Lysis Buffer:
10 mmol/L Tris-HCl (pH 7.4)
120 mmol/L KCl
20 mmol/L NaCl
0.1 % Triton X-100
0.1 mmol/L PMSF with 2 mmol/L CaCl2
8. Repeatly pass the suspension gently through a No. 25 hypodermic needle approximately 10 times or use Dounce homogenizer to completely lyse the cells (to avoid air bubbles).
9. Remove the nucleus by centrifugation of the disrupted cells at 400 g for 4 min at 4 ° C. Transfer the supernatant to a clean centrifuge tube.
10. Centrifuge the supernatant at 3000 g for 5 minutes at 4 °C to concentrate the chromosomes.
3. Separation of medium-term chromatin by hexanediol method
Reagents, kits : hexanediol buffer
Instruments, consumables : Dounce homogenizer
Experimental steps :
500 ml of suspended or monolayer cultured cells were induced to metaphase repression as described in steps 1 and 2 of the polyamine method.
2. The cells were centrifuged at 1000 r/min for 10 minutes at 4 ° C and resuspended in 10 ml of medium.
3. Cool the resuspended cells to 4 ° C for 20 minutes.
4. The cells were centrifuged at 1000 r/min for 4 minutes at 4 ° C and the medium was removed.
5. Wash the cells with hexanediol buffer at 4 ° C and centrifuge in step 4.
Hexanediol buffer:
1.0 mol/L hexanediol (2-methyl-2,4 pentanediol)
0.5 mmol/L CaCl2
0.1 mmol/L PIPES, pH 6.5
6. Resuspend the cells gently with 10 ml of cold hexane buffer and incubate for 10 minutes at 37 °C.
7. Slowly homogenize the cell suspension through a 25 gauge needle syringe or a Dounce homogenizer 10 times (to avoid excessive air bubbles).
8. The crude chromosome suspension was concentrated using a JA-20 rotor (Beclanan J-21 centrifuge) at 5000 °C/min for 15 minutes at 4 °C. Â
4. Sucrose gradient purification method
Experimental material : crude chromatin
Reagents, kits : polycarbonate centrifuge tubes
Instruments, consumables : Dounce homogenizer
Experimental steps :
1. Prepare two 18ml sucrose separation buffers (polyamine, aqueous or hexanediol buffer) at 20% and 60%, respectively, and then add them to a 50 ml polycarbonate in a linear form. In the tube, a gradient generator is used to form a density gradient along the centrifuge tube.
2. Gently apply the crude chromatin solution to the gradient and centrifuge at 2500 g for 15 minutes using a JS-13 (Beckman Instruments) or HB-4 (Sorvall) centrifuge with a bucket rotor.
3. 3 ml - part gently remove the sample from the top of the gradient and take a small amount to check for the presence of chromatin using a phase contrast microscope.
4. Collect the chromosome-containing fractions and centrifuge at 3000 g for 10 minutes.
5. Resuspend the chromosome with an isolation buffer approximately 1/30 the volume of the starting chromosomal solution and proceed to the next study.
Five, Percoll gradient method
Experimental material: chromatin
Reagents, kits : spermine, sperm, Percoll, solution
Instruments, consumables : polycarbonate centrifuge tube
Experimental steps :
1. Add spermine and spermatozoa to a final concentration of 0.8 mmol/L and 2 mmol/L in a chromatin separated from 10 ml.
2. Add 10 ml of Percoll solution and homogenize the chromatin.
Percoll solution:
89% (v/v) Percoll
5 mmol/L Tris-HCl (pH 7.4)
2 mmol/L EDTA
0.8 mmol/L spermine
2 mmol/L Jingmi
0.1% digitonin
3. Add 15 ml of Percoll solution to the homogenate and mix well.
4. Add 35 ml of Percoll mixture to a 50 ml polycarbonate centrifuge tube and centrifuge at 20,000 rpm for 30 minutes at 4 ° C using a fixed angle rotor (Beckman Instruments).
5. Aspirate all of the material above the chromosome band and recover the chromosomes in the gradient in approximately 10 ml of Percoll solution.
6. To remove the Percoll particles, dilute the chromosomes in 1:2 ratio with dilution buffer and concentrate by centrifugation at 1400 g for 45 minutes.
Sixth, glycerin gradient method
Experimental material: chromatin
Reagents, kits : glycerin, chromatin, separation buffer solution
Instruments, consumables : JS-13 rotary barrel rotor
Experimental steps :
1. Prepare 5 parts of 6 ml 10%, 20%, 30%, 40%, 50% (v/v) glycerol/chromatin separation buffer solution.
2. Chromatographic gradient sedimentation was performed using a JS-13 rotary drum rotor at 4 ° C and centrifugation at 1000 r/min for 50 minutes.
3. Collect the gradient top fraction in 3 ml portions.
4. Fix a small amount of sample (10~20 ml) on each slide on each slide and observe the chromosome-containing components by phase contrast microscopy.
5. Collect samples containing purified chromosomes.
6. The prepared sample was centrifuged at 5 ° C, 5000 r/min for 15 minutes at 4 ° C to remove glycerol, and the chromosome precipitate was resuspended in separation buffer.
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