Isolation of chloroplasts/plastids from plant tissue Materials: 1 M sucrose; 1 M

Question

Isolation of chloroplasts/plastids from plant tissue Materials: 1 M sucrose; 1 M NaHzPOs, monobasic; 1 M NazHPOs, dibasic: 5 50 rm centrifuge tubes/lab group: 1 blender/lab group; cheesecloth (1 square/lab gro up): 1 microscope/lab group: centrifuge for 50 ml, tubes that can be set at 4C; glass stir rods slides and cover slips; plastic transfer pipets; simple balance and weight boats; alumi hemocytometer/lab group; spinach and/or other plant tissues. at 4C: glass ompound num foil; 1 microscope Differential centrifugation can be used to separate cellular components, most often based on density. We will be using differential centrifugation to isolate chloroplasts from a variety of plant tissues and a hemocytometer to calculate the concentration of chloroplasts in each mL of solution. You will be mixing up your own working solution from stocks (above), so be sure to complete the calculations before coming to lab. Anyone who does not have the calculations done at the beginning of lab will have 5 points deducted from his or her lab report. 1. Make up 50 ml of Sucrose sodium phosphate buffer (0.35M sucrose plus 0.05M NaPO buffer pH 7.6.) at pH 7.6 using the three stocks provided in lab and the chart below. Place your solution on ice or in the refrigerator once you have finished. The chart below has the % of mono and di basic NaP04 solutions that should be used to make up 100 ml of 1M buffer at the given pH. Remember, you need to make 50 mM NaPOs and the solution must contain 0.35 M sucrose. PH 5.8 5.9 6.0 6.1 6.2 6.3 6.4 6.5 monobasic dibasic 92.0 90.0 87.7 85.5 81.5 77.5 73.5 68.5 62.5 56.5 51.0 45.0 39.0 33.0 28.0 23.0 19.0 16.0 13.0 10.5 10.0 12.3 15.0 19.5 22.5 26.5 31.5 37.5 43.5 49.0 55.0 61.0 67.0 72.0 77.0 81.0 84.0 87.0 89.5 6.8 7.0 7.3 7.4 7.5 7.6

Explanation / Answer

Here, first add 13 mL of the monobasic Sodium phosphate and 87 mL of disodium phosphate.

The resulting solution will be 100 mL 1M sodium phosphate buffer of pH 7.6. Now dilute this solution 10 times to produce 0.1M sodium phosphate buffer of pH 7.6

Now prepare another solution by taking 17.5 mL of 1M sucrose solution and dilute it to 25 mL to produce 25 mL of 0.7 M sucrose

Add 25 mL of 0.1M sodium phosphate buffer of pH 7.6 to the 25 mL solution of 0.7M sucrose.

The resulting solution will be 50 mL of sucrose sodium phosphate buffer(0.35M sucrose + 0.05 M NaPO4 buffer at pH 7.6)

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