789
edits
BISC110/S11: Series 3 Experiment 9 Hill Reaction: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
BISC110/S11: Series 3 Experiment 9 Hill Reaction (view source)
Revision as of 14:22, 4 April 2011
, 4 April 2011→Procedure
| Line 56: | Line 56: | ||
<br><br> | <br><br> | ||
'''Preparation of thylakoid stock solution''' | '''Preparation of thylakoid stock solution''' | ||
#Prior to lab, 60g of spinach leaves were ground with 160 mL of grinding medium in two blenders. Obtain a large blender cup containing the leaf suspension and filter half into ice-cold | #Prior to lab, 60g of spinach leaves were ground with 160 mL of grinding medium in two blenders. Two groups of four will filter the suspension from one blender while the other two groups of four will filter the suspension from the other blender. Obtain a large blender cup containing the leaf suspension and filter half of the contents into an ice-cold beaker through two layers of cheesecloth. Gently squeeze the cheesecloth to express the liquid, then discard the cheesecloth and pulp in the garbage can. Obtain a new piece of two-layered cheese cloth and filter the remaining suspension, squeezing to express the liquid and discard the cheesecloth and pulp. Now, filter half of the suspension in the beaker through four-layered cheesecloth. Squeeze and discard the cheesecloth and pulp. Filter the remaining suspension through another piece of four layered cheesecloth. Squeeze to express the liquid and discard the cheesecloth and pulp (the filtration through two layers of cheesecloth removes large debris, the filtration through four layers removes cell wall material and some nuclei). Pour half of the filtered extract into a beaker so that each group of four students has about half of the extract at their bench. [Grinding medium: 100mM Tricine NaOH pH 7.8, 400mM sorbitol, 5mM MgCl2]. '''Clean Up:''' Rinse beakers and stirrers with water immediately. | ||
#To isolate chloroplasts, divide the filtered 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). | #To isolate chloroplasts, divide the filtered 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). | ||
#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.) | #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.) | ||
