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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 10:35, 10 May 2012
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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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'''Preparation of thylakoid stock solution''' | <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! | ||
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'''Running the Hill reaction''' (work in pairs)< | <font size="+1">'''Running the Hill reaction''' (work in pairs)<font size="+1"><br> | ||
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'''Materials:''' Each pair will have a ringstand with a test tube holder, and a 150W quartz-halogen projection lamp. You will have to find the best arrangement to measure the reduction of DCPIP (loss of blue color as decrease in absorbance at wavelength 580nm) accurately and reproducibly. A light meter and timer will be provided. Set up 5 ‘reaction tubes’ to begin with, adding 5 mL of '''reaction mixture''', but do not add the thylakoid membranes, which must be kept on ice. Add these thylakoid membranes just before each reaction mixture is tested. [Reaction mix: 50 mM sodium phosphate, pH 6.8 buffer, 100 mM sorbitol, 5 mM MgCl2, 0.05 mM DCPIP]<br><br> | '''Materials:''' Each pair will have a ringstand with a test tube holder, and a 150W quartz-halogen projection lamp. You will have to find the best arrangement to measure the reduction of DCPIP (loss of blue color as decrease in absorbance at wavelength 580nm) accurately and reproducibly. A light meter and timer will be provided. Set up 5 ‘reaction tubes’ to begin with, adding 5 mL of '''reaction mixture''', but do not add the thylakoid membranes, which must be kept on ice. Add these thylakoid membranes just before each reaction mixture is tested. [Reaction mix: 50 mM sodium phosphate, pH 6.8 buffer, 100 mM sorbitol, 5 mM MgCl2, 0.05 mM DCPIP]<br><br> | ||
'''Blanking the Spectrophotometer '''([[Appendix_B/F12 | Appendix B]]): Use a tube containing | '''Blanking the Spectrophotometer ''' (Instructions are found in [[Appendix_B/F12 | Appendix B]]): Use a tube containing 50µL of your '''thylakoid suspension''' added to 5 mL of '''resuspension solution''' (NOT reaction solution!) to blank the Spec20 at 580 nm (filter level to the left). With Parafilm® over the top of the tube, invert once to mix, then wipe the tube with a Kimwipe® before blanking the instrument. Do not vortex. You may need to prepare a fresh blank periodically. | ||
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'''Varying the Reaction Rate:''' The Hill reaction occurs at different rates depending on the light intensity supplied to the thylakoids. Thylakoid concentration is also an important variable. Our goal today is to determine the best light intensity and thylakoid concentration for a 90 sec. reaction. You will measure the reaction rates by taking absorbance readings over that time period in 15 s intervals. We will use the drop in absorbance over time as a measure of reduction of blue DCPIP to colorless DCPIPH. If we can control all the variables properly, this reduction rate is also a measurement of the rate of photosynthesis. <br><br> | '''Varying the Reaction Rate:''' The Hill reaction occurs at different rates depending on the light intensity supplied to the thylakoids. Thylakoid concentration is also an important variable. Our goal today is to determine the best light intensity and thylakoid concentration for a 90 sec. reaction. You will measure the reaction rates by taking absorbance readings over that time period in 15 s intervals. We will use the drop in absorbance over time as a measure of reduction of blue DCPIP to colorless DCPIPH. If we can control all the variables properly, this reduction rate is also a measurement of the rate of photosynthesis. <br><br> | ||
Depending on the number, distribution (clumped or not) and condition of thylakoids in your reaction tube, you may obtain varying reaction rates with a given light intensity. Therefore, you will test your thylakoids neat (undiluted) and diluted 1/2 with resuspension solution. We measure the incident light using a light meter in units of µmol photons m-2 s-1. <br><br> | Depending on the number, distribution (clumped or not) and condition of thylakoids in your reaction tube, you may obtain varying reaction rates with a given light intensity. Therefore, you will test your thylakoids neat (undiluted) and diluted 1/2 with resuspension solution. We measure the incident light using a light meter in units of µmol photons m-2 s-1. <br><br> | ||
