Working with one tube at a time, do

Working with one tube at a time, do the following:
a. Label the tube with the temperature being used
b.
Add the TMB
, mix the tube, and put the tube in the appropriate water bath.
Check the
temperature of the water with a thermometer, and record this temperature on the data
sheet
; allow 10 minutes for the contents of the tube to come to equilibrium at the
desired temperature.
c. When the contents of the tube reach the correct temperature:
1)
add the enzyme
,
mix well; quickly read A
655
—this gives you a 0.00 min reading.
You do not need to "zero" the machine as long as the zero knob remains
untouched throughout the experiment.
2)
add the H
2
O
2
, mix well (and quickly)
, immediately
return to the correct water
bath.
3) allow the reaction mixture to remain in the water bath as long as possible, but you
must
have the tube in the spectrophotometer and ready
to read A
655
at
exactly
the
2:00 min time point