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BISC110/F12: Series 3 Experiment 9 Hill Reaction: Difference between revisions
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BISC110/F12: Series 3 Experiment 9 Hill Reaction (view source)
Revision as of 10:36, 10 May 2012
, 10 May 2012→Procedure
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Be sure to keep all beakers and buffers on ice. Note that there are four different media used to prepare a thylakoid suspension: grinding medium (100 mM Tricine NaOH pH 7.8, 400 mM sorbitol, 5 mM MgCl2), breaking medium (20 mM Tricine NaOH pH 7.8, 5 mM MgCl<sub>2</sub>), resuspension medium (50 mM , 100 mM sorbitol, 5 mM MgCl<sub>2</sub>)and reaction solution (100 mM sorbitol, 5 mM MgCl<sub>2</sub>, 50 mM NaPO<sub>4</sub>, 0.05 mM DCPIP). Note the differences among them. Be sure to use the correct solution. Read labels carefully throughout the experiment. | Be sure to keep all beakers and buffers on ice. Note that there are four different media used to prepare a thylakoid suspension: grinding medium (100 mM Tricine NaOH pH 7.8, 400 mM sorbitol, 5 mM MgCl2), breaking medium (20 mM Tricine NaOH pH 7.8, 5 mM MgCl<sub>2</sub>), resuspension medium (50 mM , 100 mM sorbitol, 5 mM MgCl<sub>2</sub>)and reaction solution (100 mM sorbitol, 5 mM MgCl<sub>2</sub>, 50 mM NaPO<sub>4</sub>, 0.05 mM DCPIP). Note the differences among them. Be sure to use the correct solution. Read labels carefully throughout the experiment. | ||
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<font size="+1">'''Preparation of thylakoid stock solution'''<font size="+1"> | <font size="+1">'''Preparation of thylakoid stock solution'''</font size="+1"> | ||
#Work in groups of eight students in steps 1 and 2. Throughout the procedure, be sure to wash glassware with water and the round bottom centrifuge tubes that have come in contact with chlorophyll using 70% ethanol. Please do this as soon as you have finished using this equipment. | #Work in groups of eight students in steps 1 and 2. Throughout the procedure, be sure to wash glassware with water and the round bottom centrifuge tubes that have come in contact with chlorophyll using 70% ethanol. Please do this as soon as you have finished using this equipment. | ||
#Prior to lab, 60 g of spinach leaves were mechanically macerated our lab specialists with 160 mL of grinding medium using Waring blenders. The ground up spinach will be given to you in a 500ml iced cold beaker. A group of 8 students will filter the spinach through first 2 layers of cheese cloth into another iced cold 500 ml beaker. You must gently squeeze the spinach in the cheese cloth to express the liquid and leave the pulp and debris in the cheesecloth. Discard the cheesecloth with the pulp in the trash can. Filter the expressed liquid (containing your intact cholorophasts) through 8 layers of cheesecloth into another 500ml cold beaker. Gently squeeze the cheesecloth to express the refiltered liquid, then discard the cheesecloth and pulp in the trash can. '''Clean Up:''' Rinse beakers with water immediately BUT DO NOT DISCARD THE FILTERED EXTRACT to be used in the next step! | #Prior to lab, 60 g of spinach leaves were mechanically macerated our lab specialists with 160 mL of grinding medium using Waring blenders. The ground up spinach will be given to you in a 500ml iced cold beaker. A group of 8 students will filter the spinach through first 2 layers of cheese cloth into another iced cold 500 ml beaker. You must gently squeeze the spinach in the cheese cloth to express the liquid and leave the pulp and debris in the cheesecloth. Discard the cheesecloth with the pulp in the trash can. Filter the expressed liquid (containing your intact cholorophasts) through 8 layers of cheesecloth into another 500ml cold beaker. Gently squeeze the cheesecloth to express the refiltered liquid, then discard the cheesecloth and pulp in the trash can. '''Clean Up:''' Rinse beakers with water immediately BUT DO NOT DISCARD THE FILTERED EXTRACT to be used in the next step! | ||
