How?
Why?
Photosynthesis &
e transport
Pathway thinking
X1
X2
Following the Scientific Approach: Predicting
Alteration
•
On page 13-10, Step 6, change the target absorbance to 0.1
instead of the 0.2 specified in the manual
•
Use about 4 spinach leaves, no need to weigh out 4g
•
Spin extract in centrifuge at 3 min first spin and 5 min second
spin (we do not have fast and slow centrifuges) keep on ice
until ready to combine pellets.
•
Keep final chloroplast solution on ice until ready for further
procedural items!! Ice bucket is fine for this
Technical aspects I
•
Where’s the photosynthetic stuff?
•
Round I: get rid of huge chunks of
cellular debris, keep supernatant
•
Round II: acquire chloroplasts,
keep pellet
Technical aspects II
•
When will dinner be ready?
•
At what point in time will the chloroplast-induced reduction
have taken place, but the random reduction not yet have
occurred at significant levels?
•
Record absorbance values at different time intervals during
actual experiment
Technical aspects
III
•
What does herbicide need to achieve to ‘do its thing’?
•
What’s happening to DCPIP sitting in the tube prior to the
experiment starting?
•
What happens to results if tubes are not thoroughly mixed?
•
Is there a difference between 600 & 660 nm? Should you pay
attention to which the protocol calls for when?
•
If you have a tube that’s supposed to be ‘dark’, where should it be
most/all of the time?
Technical Aspects IV
Your chloroplast
preparation
Dilute
1 into
acetone
3
2
Read at 660
Compare reading
to 0.1
Dilute the original
material by the ratio
(actual reading/0.1)
Apply your
calculated dilution
to the preparation
(dilute using
buffer, not
acetone!!!)
Your experimental
material!
In other words…
•
First dilute extract solution in acetone per manual specifications
•
Find out the dilution needed to obtain absorbance reading of 0.1 on
spec at 660nm
•
Then, dilute ACTUAL extract used for experiment by amount
determined from spec reading at 660nm to get to 0.1. You do not need
to do the initial dilution used with acetone, only the dilution needed to
reduce to 0.1 absorption. Ex.
•
Dilute ACTUAL extract used for experiment with sodium phosphate
buffer or the leaves will die!
Thinking thoughts
•
Why is acetone used?
•
Why do you think the stock chloroplast solution must be diluted?
•
Why is the chlorophyll concentration an important factor?
Additional Important Points
•
Absorbance readings of actual experiment should be done at
600nm, you can change the spec wavelength at your station this
week just don’t forget to!
•
Make sure to add DCPIP last to all tubes, remember light could
be a contributing factor
•
Add all components to tubes in correct amounts first, add
DCPIP last (make sure they all add up to 5mL)
•
Suggested: Water, buffer, herbicide first, extract second (keep
chilled until needed), DCPIP last (keep in dark until needed)
Additional Important Points
•
Start all tubes under the lights at the same time! The one in
the dark can be covered in foil or kept in a drawer
•
ALL tubes must be measured for absorbance every two
minutes! Don’t forget a zero min absorbance reading
•
Can stop absorbance readings once reducing solutions stay
at/around the same absorbance for more than 4 minutes (two
readings)
More thoughts
•
Where should your tubes be relative to the lights?
•
Should you allow your contents to settle to the bottom of the
tube?
•
Is it advantageous to have the tubes vertical this week?
Reports: Don’t Forget
To Include…
•
Figure: Indicate both hypothesis on figure
•
Predictions: For each tube! What is the expected outcome?
•
Table: Purposes for each tube, why are you testing each tube and
what will it prove? Why is it an experimental tube or a control?
•
Conclusions: why do your observations/results of each control tube
support or reject the hypotheses proposed or the outcome of the
experimental tube?
Homework
Before 12/6