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BISC110: Series 3 Experiment 9 Hill Reaction: Difference between revisions
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BISC110: Series 3 Experiment 9 Hill Reaction (view source)
Revision as of 13:18, 9 November 2009
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1. Working in groups of 4 students at a bench, remove large stems and veins and unhealthy parts and then weigh 30g of spinach leaves. Tear leaves into 1-inch-wide strips. In a chilled blender, grind leaf tissue with 80mL ice-cold grinding medium for 5–10s at high speed. Filter the suspension into ice-cold beakers first through two, then eight layers of cheesecloth (the first filtration removes large debris, the second removes cell wall material and some nuclei). Gently squeeze the cheesecloth to express the liquid in both steps, then discard the cheesecloth and pulp in the garbage can. [Grinding medium: 100mM Tricine NaOH pH 7.8, 400mM sorbitol, 5mM MgCl2] | 1. Working in groups of 4 students at a bench, remove large stems and veins and unhealthy parts and then weigh 30g of spinach leaves. Tear leaves into 1-inch-wide strips. In a chilled blender, grind leaf tissue with '''80mL ice-cold grinding medium''' for 5–10s at high speed. Filter the suspension into ice-cold beakers first through two, then eight layers of cheesecloth (the first filtration removes large debris, the second removes cell wall material and some nuclei). Gently squeeze the cheesecloth to express the liquid in both steps, then discard the cheesecloth and pulp in the garbage can. [Grinding medium: 100mM Tricine NaOH pH 7.8, 400mM sorbitol, 5mM MgCl2] | ||
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Clean Up: Rinse blender jar parts, beakers and stirrers with water immediately. | Clean Up: Rinse blender jar parts, beakers and stirrers with water immediately. | ||
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2. To isolate chloroplasts, divide the extract into two equal portions and pour them into two 50mL plastic round-bottom, capless centrifuge tubes. Balance the two tubes by transferring extract from the heavier tube to the lighter one, and centrifuge at 1000 x g in the SS34 rotor (see the conversion chart for RPMs) for 5min in the refrigerated (4ºC) centrifuge. After the spin, each pair of students continues with one of these tubes. From now on you are working in pairs. Carefully decant (pour off) the pale green supernatant and discard it. Save the green pellet (chloroplast-enriched fraction). | 2. To isolate chloroplasts, divide the extract into two equal portions and pour them into two 50mL plastic round-bottom, capless centrifuge tubes. Balance the two tubes by transferring extract from the heavier tube to the lighter one, and centrifuge at 1000 x g in the SS34 rotor (see the conversion chart for RPMs) for 5min in the refrigerated (4ºC) centrifuge. After the spin, each pair of students continues with one of these tubes. From now on you are working in pairs. Carefully decant (pour off) the pale green supernatant and discard it. Save the green pellet (chloroplast-enriched fraction). | ||
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3. Using a glass rod, gently resuspend the pellets by mixing 1–2mL of breaking medium into the pellet. Make sure the pellet is completely detached from the wall and mixed into the resuspension. Avoid air bubbles (O2) that may oxidize enzymes and thereby reduce activity. The breaking medium is intended to shock the chloroplasts osmotically, thereby breaking open the organelles' outer membranes and releasing the stroma while leaving the thylakoid membranes intact. (Breaking medium: 20mM Tricine NaOH pH 7.8, 5mM MgCl2. Note the absence of sorbitol.) | 3. Using a glass rod, gently resuspend the pellets by mixing 1–2mL of '''breaking medium''' into the pellet. Make sure the pellet is completely detached from the wall and mixed into the resuspension. Avoid air bubbles (O2) that may oxidize enzymes and thereby reduce activity. The breaking medium is intended to shock the chloroplasts osmotically, thereby breaking open the organelles' outer membranes and releasing the stroma while leaving the thylakoid membranes intact. (Breaking medium: 20mM Tricine NaOH pH 7.8, 5mM MgCl2. Note the absence of sorbitol.) | ||
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4. Bring the resuspended pellets to a volume of about 25mL with cold breaking medium (50mL centrifuge tube half-full), balance against a tube of water, and centrifuge at 1900 x g for 5min. Discard the supernatant. Resuspend the resulting pellet in 1.5mL of resuspension medium. This suspension is your stock preparation of thylakoids to be used in the Hill reaction. Keep it on ice. (Resuspension medium: 50mM Tricine NaOH pH 7.8, 100mM sorbitol, 5mM MgCl2). | 4. Bring the resuspended pellets to a volume of about 25mL with '''cold breaking medium''' (50mL centrifuge tube half-full), balance against a tube of water, and centrifuge at 1900 x g for 5min. Discard the supernatant. | ||
5. Resuspend the resulting pellet in 1.5mL of '''resuspension medium'''. This suspension is your stock preparation of thylakoids to be used in the Hill reaction. Keep it on ice. (Resuspension medium: 50mM Tricine NaOH pH 7.8, 100mM sorbitol, 5mM MgCl2). | |||
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