Bio-Rad Biofuel Enzyme Kit User Manual
Quick Guide
Activity 1: Determine the Reaction
Rate in the Presence or Absence
of an Enzyme
1. Find your 15 ml conical tubes labeled
“Stop Solution”, “1.5 mM Substrate”,
“Enzyme” and “Buffer”. Write your initials
on each tube.
2. Label five cuvettes E1–E5.
3. Label the two remaining cuvettes “Start”
and “End”.
4. Using a clean DPTP, pipet 500 µl of stop
solution into each labeled cuvette. Rinse
the DPTP well with water.
5. Label one empty 15 ml conical tube
“Enzyme Reaction” and the other
“Control”.
6. Using a clean DPTP, pipet 2 ml of 1.5 mM
substrate into the 15 ml conical tube
labeled “Enzyme Reaction”. Use the
same DPTP and pipet 1 ml of 1.5 mM
substrate into the conical tube labeled
“Control”. Rinse the DPTP well with
water.
7. Label one DPTP “E” for enzyme and the
other “C” for control. Only use the DPTP
labeled “E” for the enzyme reaction tube
and the DPTP labeled “C” for the control
reaction tube.
26
Quick Guide
QUICK GUIDE
Label up here
Stop Solution
1.5 mM Substrate ControlEnzyme reaction
Start
End
E1 E2
E3
E4 E5
Read and understand steps 8–11 fully
before proceeding. These steps are time
sensitive!
8. Using the DPTP labeled “C”, pipet 500 µl
of buffer into the 15 ml conical tube
labeled “Control” and gently mix. Once
you have mixed the buffer with the
substrate, remove 500 µl of this solution
and add it to your cuvette labeled “Start”.
9. Using the DPTP labeled “E”, pipet 1 ml of
enzyme into the 15 ml conical tube
labeled “Enzyme Reaction”. Gently mix,
then START YOUR TIMER.
10. At the times indicated, use the DPTP
labeled “E” to remove 500 µl of the
solution from the “Enzyme Reaction”
tube and add it to the appropriately
labeled cuvette containing the stop
solution.
11. After all the enzyme samples have been
collected, use the DPTP labeled “C” to
remove 500 µl of the solution from the
“Control” reaction tube and add it to the
cuvette labeled “End”.
12. Proceed with the analysis of your
samples. After you have finished your
analysis, rinse out your reaction (conical)
tubes, cuvettes, and DPTPs with copious
water and save them for later activities.
Note: Do not discard unused stock solutions.
They will be used for the next activity.
27
Quick Guide
QUICK GUIDE
Enzyme
reaction
E1
1 min
E2
2 min
E3
4 min
E4
6 min
E5
8 min
Control
End
8 min
Buffer Control
Enzyme Enzyme reaction
Control Start
Quick Guide
Activity 2: Determine the Effect of
Temperature on the Reaction Rate
1. Label your cuvettes “0°C”, “22°C”, and
“37°C”.
2. Using a clean DPTP, pipet 500 µl of stop
solution into each cuvette. Wash the
DPTP out thoroughly with water.
3. Label three 1.5 ml microcentrifuge tubes
“0°C Enzyme”, “22°C Enzyme”, and
“37°C Enzyme”. Using a clean DPTP,
pipet 250 µl of enzyme into each
microcentrifuge tube. Rinse out the
DPTP thoroughly with water.
4. Label three 1.5 ml microcentrifuge tubes
“0°C Substrate”, “22°C Substrate”, and
“37°C Substrate”. Using a clean DPTP,
pipet 500 µl of 1.5 mM substrate into
each microcentrifuge tube. Rinse out the
DPTP thoroughly with water.
5. Place the tubes labeled “0°C Enzyme”
and “0°C Substrate” in the ice cup. Place
the tubes labeled “22°C Enzyme” and
“22°C Substrate” on your lab bench.
Place the tubes labeled “37°C Enzyme”
and “37°C Substrate” in the beaker of
warm water at 37°C. Allow the tubes to
equilibrate to their respective temperatures for at least 5 minutes.
28
QUICK GUIDE
Quick Guide
Label up here
Stop Solution
0°C
22°C 37°C
Enzyme
0°C
Enzyme
22°C
Enzyme
37°C
Enzyme
1.5 mM Substrate
0°C
Substrate
0°C
Substrate
22°C
Substrate
37°C
Substrate
0°C
Enzyme
0°C
22°C
Enzyme
Room
Temperature
37°C
Enzyme
37°C
22°C
Substrate
37°C
Substrate
6. Have a stopwatch ready. Using a clean
DPTP, pipet the 250 µl of enzyme from
the tube labeled “0°C Enzyme” into the
tube labeled “0°C Substrate”, and place
the tube now containing your enzyme
and substrate mix back on ice. Add the
22°C enzyme to the 22°C substrate
solution, and place that tube back on the
lab bench. Add the 37°C substrate to the
37°C enzyme solutions, and put that tube
back into the 37°C water bath. START
YOUR TIMER.
7. After 2 minutes, use a clean DPTP for
each temperature reaction to transfer
500 µl of your reaction to the appropriately
labeled cuvette containing the stop
solution.
8. Proceed with the analysis of your
samples. After you have finished your
analysis, rinse out the cuvettes and
DPTPs with copious water and save
them for later activities.
Note: Do not discard unused stock solutions.
They will be used for the next activity.
29
Quick Guide
QUICK GUIDE
0°C
0°C S
22°C S 37°C S
0°C E
0°C
22°C E
Room
Temperature
37°C E
37°C
0°C
S+E
22°C22°C
S+E
37°C
37°C
S+E
2 min
Loading...