Experiments on the graded separation of cell nuclei
Experiments on the graded separation of cell nuclei
Fractionation (Fractionation) is a gradual sedimentation by centrifugation from low to high speed. First use low speed to make the larger particles precipitate, and then use a higher speed, will float in the supernatant particles precipitate down, so that a variety of cellular structures, such as the nucleus, mitochondria, etc. can be separated.
Operation method
basic program
Materials and Instruments
Mice Move 1. After the mice were executed by cervical dislocation, the abdomen was quickly dissected to remove the liver, which was cut into small pieces (removing connective tissue) and placed as soon as possible in a beaker containing 0.9% NaCl, washed repeatedly to remove as much blood as possible, and the liquid on the surface was removed by suction with filter paper.2. Place approximately 1 g of wet weight liver tissue in a small flat dish and measure 8 ml of pre-cooled 0.25 mol/L sucrose-0.003 mol/L calcium chloride solution with a measuring cylinder, add a small amount of the solution to the dish and shear the liver tissue as much as possible before adding it all.3. Pour the chopped liver tissue into the homogenizing tube, so that the lower end of the homogenizer is immersed in a vessel containing ice, hold it in the left hand, insert the homogenizing rod vertically into the tube with the right hand, rotate it up and down and grind it for 3-5 times, filter the homogenized liquid into the centrifugal tube with 3 layers of gauze, then prepare a smear ①, mark it well, and dry it naturally.4. The centrifuge tube containing the filtrate is flattened and placed in a regular centrifuge and centrifuged at 2500 rpm for 15 minutes. For more product details, please visit Aladdin Scientific website.
Sucrose Calcium chloride solution Toluidine blue stain Janus green B stain NaCl solution
Glass homogenizer Centrifuge Balance Optical microscope Slide Coverslip Centrifuge Tube Dropper Measuring cylinder Beaker Glass funnel Dissecting scissors Tweezers
(1) Remove the supernatant slowly, transfer it to a high-speed centrifuge tube, and store it in a beaker with ice for use in isolating mitochondria.
(2) At the same time, apply a piece of supernatant ② Mark it well and dry it naturally.
(3) The remaining precipitate is subjected to the next step.5. Suspend the precipitate with 6 ml of 0.25 mol/L sucrose-0.003 mol/L calcium chloride solution, centrifuge at 2500 rpm for 15 minutes and discard the supernatant, blow the residual liquid with a pipette to form a suspension, place a drop on a clean slide, smear ③, and dry naturally.6. Stain the ①, ② and ③ smears with 1% toluidine blue and cover the smears for observation.