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Isolation of Chloroplasts From Spinach Leaves

1. The document describes a protocol for isolating chloroplasts from spinach leaves through a process of grinding, filtering, and centrifuging the leaf tissue. 2. The protocol involves preparing solutions, chopping spinach leaves, grinding the chopped leaves, filtering and centrifuging the ground tissue to pellet chloroplasts, and resuspending the chloroplasts in a suspension solution. 3. A separate section then describes determining the chlorophyll content of the isolated chloroplasts through spectrophotometric analysis of acetone extracts of the chloroplast suspension.

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Sadia Sharmin Nilima
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1K views3 pages

Isolation of Chloroplasts From Spinach Leaves

1. The document describes a protocol for isolating chloroplasts from spinach leaves through a process of grinding, filtering, and centrifuging the leaf tissue. 2. The protocol involves preparing solutions, chopping spinach leaves, grinding the chopped leaves, filtering and centrifuging the ground tissue to pellet chloroplasts, and resuspending the chloroplasts in a suspension solution. 3. A separate section then describes determining the chlorophyll content of the isolated chloroplasts through spectrophotometric analysis of acetone extracts of the chloroplast suspension.

Uploaded by

Sadia Sharmin Nilima
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Isolation of Chloroplasts from Spinach Leaves Contributor: The Laboratory of William Heidcamp at Gustavus Adolphus University

Overview This protocol describes a simple procedure for isolating chloroplasts from spinach leaves. Procedure 1. Prepare an ice bath and pre-cool all glassware to be used (including a mortar and pestle). 2. Select several fresh spinach leaves. Remove the large veins by tearing them loose from the leaves. Weigh out 4 g of de-veined leaf tissue. 3. Chop the tissue as fine as possible with a knife and chopping board. 4. Add the tissue to an ice-cold mortar containing 15 ml of Grinding Solution and grind the tissue to a paste. 5. Filter the ground up tissue solution through double layered cheesecloth into a beaker and squeeze the tissue pulp to recover all of the liquid suspension. 6. Transfer the green suspension to a cold 50 ml centrifuge tube and centrifuge for 1 minute at 200 X g at 4C to pellet the unbroken cells and cell fragments. 7. Decant the supernatant into a clean centrifuge tube and centrifuge again for 7 minutes at 1,000 X g to pellet the chloroplasts. Decant and discard the supernatant. 8. Resuspend the chloroplasts in 5 ml of cold Suspension Solution (or 0.35 M NaCl). Use a cold glass stirring rod to gently disrupt the packed pellet. 9. Enclose the tube in aluminum foil and place at 0 to 4C (see Hint #1). 10. Using a hemacytometer, determine the number of chloroplasts per ml of suspension media. 11. To determine the chlorophyll content see Protocol on Determination

of Chlorophyll Content in Chloroplasts. Solutions Grinding Solution 0.33 M Sorbitol pH 6.5 Adjust with HCl 4 mM MgCl2 2 mM Ascorbic Acid (Vitamin C) 10 mM Sodium Pyrophosphate

0.35 M NaCl Spinach Leaves (Fresh) Suspension Solution

0.33 M Sorbitol pH 7.6 Adjust with NaOH 1 mM MgCl2 50 mM HEPES 2 mM EDTA

BioReagents and Chemicals Spinach Leaves (Fresh) Magnesium Chloride Ascorbic Acid Sorbitol HEPES Cheesecloth Hydrochloric Acid EDTA Sodium Hydroxide Sodium Pyrophosphate Sodium Chloride

Determination of Chlorophyll Content in Chloroplasts Contributor: The Laboratory of William Heidcamp at Gustavus Adolphus University

Overview This protocol describes how to determine the chlorophyll content of chloroplasts using a spectrophotometric assay. Procedure 1. Isolate Chloroplasts from spinach leaves (See protocol for preparation

of chloroplasts from spinach leaves). 2. Pipet 1 ml of a chloroplast suspension into a 15 ml centrifuge tube and add 8 ml of Acetone. Mix thoroughly. 3. Add 1 ml of ddH2O, mix again and centrifuge at 1,000 X g for 3 to 5 minutes. 4. Measure the absorbance of the sample from Step 3 on a spectrophotometer at 652 nm (See Hint #1). 5. Calculate the amount of total chlorophyll in 1 ml of the Chloroplast suspension. The amount of chlorophyll is given by the equation: mg chlorophyll per ml = Absorbance / 34.5

BioReagents and Chemicals Acetone

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