Perfect pipetting practice perpetuates prideful performace!
NORMAL PIPETTES
You have already used regular pipettes in honors biology. We have 1 mL , 5 mL, and 10 mL pipettes and
two different types of pipette pumps. I have green pipette pumps (used with 5 mL and 10 mL pipettes) and
blue pipette pumps (used with 1 mL pipettes).
Helpful hints . . .
 Start with the plunger slightly up. This will allow you to completely dispense the liquid into the test
tube.
 Slowly withdraw the liquid making sure that the liquid does not go all the way up to the cotton in
the pipette. If the cotton gets wet, the pipette will be nonfunctional!
 Choose the appropriate pump for the pipette size. I think of Green Giant (larger pipettes -- 5 mL
and 10 mL) & Blue smurf (smaller pipettes – 1 mL)
 Read the bottom of the meniscus
Use your “normal” pipettes to set up the following test tubes (large test tubes – not cuvettes). Each
student should set up one test tube themselves.
Large Test Tube 1
Large Test Tube 2
0.50 mL of H2O with 1 mL pipette 1.00 mL of H2O with 1mL pipette
4.5 mL of H2O with 5 mL pipette 1.5 mL of H2O with 5 mL pipette
5.0 mL of H2O with 10 mL pipette 7.5 mL of H2O with 10 mL pipette
Now pour all of the contents of Large Test Tube 1 into the graduated cylinder. You should have 10 mL of
water. How did you do? circle one ( over pipette, perfect pipette, under pipette )
Now pour all of the content of Large Test Tube 2 into the graduated cylinder. You should once again have
10 mL water. How did you do? circle one ( over pipette, perfect pipette, under pipette )
MICROPIPETTES
Micropipettes are used to measure small volumes of liquids very accurately and precisely. These are called
micropipettes because they measure volumes in μL (microliters).
1000 μL = 1.000 mL.
So 250 μL is 0.250 mL.
PROPER USE OF DIGITAL MICROPIPETS
1. Never attempt to use the pipette without the tip in place as fluid would be sucked into the piston,
destroying the pipette.
2. Never lay down a pipette with a filled tip as fluid will run back into the piston.
3. Never let the plunger snap back after withdrawing or ejecting fluid.
4. Never go beyond the limitations of the pipette (100-1000 L or 1-10 L)
Micropipeting Procedure:
1. Rotate button to set the desired volume.
2. Push pipette firmly into the proper size tip. Do not touch the tip (sterile technique).
3. Pick up reagent tube from which you will be withdrawing. Hold it at eye level. You must watch as you
pipette.
4. Hold the pipette vertical!!!!!! Depress the plunger to the first stop, and hold it in position.
5. Dip the tip into the solution to be pipetted, and draw fluid into the tip by gradually releasing the plunger.
Make sure you account for the change in liquid level in reagent tube – it will decrease in level!!
6. Slide the tip along the inside wall of the reagent tube to dislodge any excess fluid anchoring to the
outside of the tip. (only do this when using microcentrifuge tube)
7. Touch the pipette tip to the inside wall of the microcentrifuge tube into which you want to empty the
sample. This action creates a capillary effect which helps draw fluid out of the tip.
8. To expel the sample, slowly depress the plunger to the first stop. Now press on to the second stop to
blow out the last bit of fluid.
9. Hold the plunger down and slide the pipette out of the reagent tube. If you fail to hold the plunger
down you could easily suck your sample back into your tip and never know it!!!
10. Eject the tip into the waste beaker by depressing the eject button (closest to numbers).
11. The hints below will prevent contamination of your reagents.
 Add a single reagent sequentially to all reaction tubes.
 Release each reagent drop onto a new location on the inside wall of the microcentrifuge tube. This
saves tips because you can use the same tip to pipette reagent into each microcentrifuge tube.
 Use a fresh tip for each new reagent you pipette (only if you are using different reagents)
 In the enzyme lab – you are only going to use the micropipette for measuring the enzyme. You can
use the same tip for the whole lab.
MICROPIPET EXERCISE
1. Label two 1.5 mL microcentrifuge tubes A and B. Assign one person / per tube (A/B)
2. Us the matrix below to use as a checklist as you add solutions 1 thru 4 to each reaction tube.
TUBE
A
B
SOL #1
354 L
150 L
SOL #2
216L
250 L
SOL #3
220 L
301 L
SOL #4
110 L
199 L
3. Set the 100- 1000 L micropipette to add appropriate volumes of solutions 1-4 to tubes A and B.
4. You measured a total of 900 L of reactants to each test tube. Now, with the help of your partner, grab
your 1 mL pipette and slowly pull the liquid from the microcentrifuge tube into the 1 mL “normal” pipette
until the liquid is gone from the microcentrifuge tube.
How did you do with tube A? circle one ( over pipette,
perfect pipette, under pipette )
How did you do with tube B? circle one ( over pipette,
perfect pipette, under pipette )
Remember – 900 μL is ____________ mL.
Place the solution back into the microcentrifuge tube. Rinse out the microcentrifuge tube so we can reuse
it next period.
Spectrophotometer Fun!
The spectrophotometer allows us to quantify color change. It allows the user to decide what wavelength of
light passes through a liquid-filled cuvette. The cuvette is a specially ground test tube that is consistent in
width and optical quality. The other side of the spectrophotometer has a sensor that analyzes the amount
of the light that passes through the cuvette. The user can measure transmittance (amount of light that
goes through the liquid) or absorbance (amount of light that is absorbed by the liquid).
Here is a diagram of a spectrophotometer.
(Cuvette)
Here is a picture of a spectrophotometer.
Set mode to Absorbance
Sample Chamber
Wavelength Selector
Wavelength Adjust
lever
Set Absorbance
to Infinity &
Power Switch
After Blank is in
chamber set to
zero Abosrbance
1.
2.
3.
4.
5.
Use the Power switch to turn the machine on. Let I warm up 5 minutes before taking readings
Make sure that the wavelength adjust lever is in the correct range
Use wavelength selector knob to adjust to the correct wavelength (435 nm)
Make sure the mode is set to Absorbance
Adjust the power switch until the digital readout is blinking 1.999 or the analogue absorbance
reading is infinity (DON’T TOUCH THIS KNOB AGAIN DAGNABIT!!!!!!)
6. Kimwipe the blank cuvette and then place the blank in the sample chamber
7. Turn after sample adjustment knob until readout is 0 absorbance (you have just blanked the
machine) --- aka -- “tricked” the machine into thinking this is clear.
8. Now you can place other cuvette samples into the chamber and take absorbance readings
With your partner in crime complete the following . . .
1. Place the following in a cuvette.
( 4.5 mL of H2O using 5.0 mL pipette & 1.5 mL apple juice using 1.0 mL pipette )
2. Now follow steps 1-7 above using this as the blank.
3. After you blank the spectrophotometer , add 250 μL of Red solution to you blank cuvette. Place
your thumb over the top of the cuvette and slowly invert the tube. Once the solution is mixed,
Kimwipe, and then place it in the chamber.
What is the absorbance?_______________________
be similar because you measured the same amounts.
. Check the other groups absorbance – they should
4. Add another 250 μL of Red solution to the same cuvette, invert, Kimwipe and place it in the
chamber.
What is the absorbance?_______________________. Once again check with the other group.
5. Add 250 μL one last time. Mix, Kimwipe and record absorbance.
What is the absorbance?_______________________. Once again, check with the other group.
A couple O’ Questions
Why did we set the wavelength to 435 nm in this exercise?
If we blanked the spectrophotometer with only water instead of water/apple juice, would your absorbance
reading be the same? Why / Why not.
If we were testing the absorbance of a blue solution instead of a red solution, what wavelength would you
set your spectrophotometer? Explain.