PROC FOR QUES 3 - TEMP

PROC FOR QUES 3 - TEMP - PROCEDURE FORQUESTION 3: EFFECT...

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Unformatted text preview: PROCEDURE FORQUESTION 3: EFFECT OF TEMPERATURE General procedural hints for groups studying the effect of temperature on turnip peroxidase: 1. You will follow the procedure you used in Exercise 6, the baseline experiment (See pages 10-11) but you will use the new Mixing Table. You will need to do a new Baseline experiment using room temperature as your baseline- Tubes 2 and 3. You will have to do another baseline because the turnip extract will be different from that made in the previous lab. The baseline run will be the positive control which contains the variable for which you are testing. You will compare all other conditions studied to the baseline condition (Tubes 2 and 3). Note that in this case Tube 1 is your "blank" as well as your "negative control." You will use Tube 1 to calibrate the spectrophotometer for each set of conditions (2 and 3), (4 and 5), (6 and 7), etc. YOU MUST HAVE A TUBE 1, FOR EACH CONDITION. ALL TUBES MUST BE INCUBATED AT THE GIVEN TEMPERATURE STUDIED. 2. As part of your protocol, you should refer to the Mixing Table for all conditions for your hypotheses/predictions. Mixing Table: Table for Effect of Temperature on the Activity of Turnip Peroxidase (all volumes in ml) Buffer - pH 5 Hydrogen Turnip Guaiacol Temperature (mL) Peroxide Extract (mL) (mL) (mL) [S] [E] Tube 1 5 mL 2 0 1 Varies Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 Tube 8 Tube 9 Tube 10 Tube 11 0 4 0 4 0 4 0 4 0 4 2 0 2 0 2 0 2 0 2 0 0 1 0 1 0 1 0 1 0 1 1 0 1 0 1 0 1 0 1 0 23 C Room Temp 4 C 37 C Body Temp 50C 100 C Boiling 3. Pipet and test 23 C, then do other temperatures. Pipet and test the contents of tubes 2 and 3 first. Then, incubate these two tubes plus tube 1. While they are incubating for 10-15 minutes, you may pipet the contents of tubes 4 and 5. Then, continue to repeat this pattern for the rest of the pair of tube combinations. 1 4. Get all your supplies including an extra beaker to bring back your tubes from the water bath after incubation. You need to try to keep the temperature as consistent as possible to the temperature that you are testing. 5. You will need to get ice for the 4 C; use 23 C for room temp; use one water bath set for 37 C, use the digital water bath set for 50 C. Make sure you check the water baths for water levels and temperature levels. [For the Precision water baths, the temperature control and high temperature control are the same as the temperature you are trying to set. There is also a digital water bath that should be used for the 50 C.] For 100 degrees Celsius, you should use a hot plate. Make sure it does not boil dry and make sure you use test tube holders to prevent burns. IMPORTANT for 100 C - ONLY BOIL ABOUT 3 ML OF THE ENZYME BY ITSELF AND THEN USE THIS IN TUBE 11. (If you boil Tubes 1 and 10, the guaiacol may breakdown at this temperature that will affect your reaction.) Incubate at least 10-15 minutes. When you are removing your test tubes after incubation, put some of the water from the water bath in a beaker along with the tubes. This will maintain the desired temperature. 6. Tables shown below for each condition can be used to collect data. For your raw data tables, you should only show the averages and standard deviations and should have a proper title. Table 1. 23 C (Baseline) Time (sec.) Run 1 0 20 40 60 80 100 120 Table 2. 4C Time (sec.) 0 20 40 60 80 100 120 Table 3. 37 Time (sec.) 0 20 40 60 80 Run 2 Run 3 Average S.D. Run 1 Run 2 Run 3 Average S.D. Run 1 Run 2 Run 3 Average S.D. 2 100 120 Table 4. 50C Time (sec.) 0 20 40 60 80 100 120 Table 5. 100C Time (sec.) 0 20 40 60 80 100 120 Run 1 Run 2 Run 3 Average S.D. Run 1 Run 2 Run 3 Average S.D. Table 6. Temperature vs. Reaction Rate Temperature 23C 4 37 50 100 Reaction Rate (abs./min.) 3 ...
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This note was uploaded on 04/07/2008 for the course BIOSCI 0050 taught by Professor Carollafarve during the Fall '07 term at Pittsburgh.

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