446/546 handout 1 Pipetting excercise
Today we will practice pipetting water and a more viscous liquid, glycerol. The goal is to
learn how to use a pipette. Accurate and reproducible volume measurements with the
pipette are critical to ensure that your experiments provide correct data. Pay attention to
your pipetting technique. It is crucial for getting good results in the lab.
Some general considerations
-What are the different pipettes?
-How are the volumes set?
-How do they operate?
Tips
- Hold the pipette vertically
- Use a smooth up and down motion
- Pre-wetting your tip improves accuracy
Pipetting exercise
1) Accurate pipetting
Use the P20 pipette, pipette tips, filter paper and colored solution
- Adjust the pipette volume to 10 μl by turning the knob near the top of the pipette.
- Place a disposable tip on the pipette. Be sure it is on tight and makes a complete seal
with the pipette.
- Depress the plunger button on top of the pipette with your thumb to the first stop.
- Dip the point of the tip under the surface of the colored water then slowly release the
plunger to its resting position to draw up the liquid, eject to first stop and repeat.
- Place the pipette tip on the filter paper.
- Slowly eject the liquid by depressing the plunger to the first stop and then further to the
second stop.
- The second stop is as far as the plunger will go and is designed to fully eject all the fluid
with an extra puff of air.
Note: When ejecting liquid from the pipette into another solution, be sure you do not
release the plunger from the second stop while the tip is still in the solution. This will
obviously suck fluid back into the pipette tip and your volumes will not be accurate.
Practice this as much as necessary to get reproducible circular spots on the filter paper.
Then make 4 spots of 3 μl, 5 μl, 10 μl, and 20 μl spots on the filter paper (total of 16
spots). Dry and save the paper, YOU NEED it for your assignment (see other side).
2) Mixing:
During labs you will need to use the pipette to mix solutions.
- Pipet 500 μl regular water in a snap cap, then dispense 10 μl blue water into the water
- Pipet 10 μl of blue liquid “as a bead” onto the inside wall of a snap cap. Then add 500
μl of regular water.
Which of the above is the more effective way to mix a large and a small volume of
liquids?
3) Using your pipette for mixing:
You can mix liquids by pipetting “up and down” several times. The pipette should never
go beyond the first stop during mixing. Simply depress the plunger to the first stop and
then release it to its resting point. Repeat the desired number of times. When you are
done mixing you will depress to the second stop to force all liquid from the tip and
remove the tip from the liquid while the plunger is depressed to prevent sucking fluid
back in.
- Pipet 500 μl regular water in a snap cap, then mix 100 μl blue water into the water,
pump up and down 5 times.
- Repeat until comfortable with this procedure
4) Viscosity
Now try your pipettes to transfer volumes of 10 μl , 100 μl and 500 μl of glycerol into a
snap cap.
Hint, GO SLOW, REALLY SLOOOOOW
Assignment: PUT YOUR WORK IN YOUR NOTE BOOK
Measure the area of each spot on your filter paper (exercise 1). You should do this by
averaging perpendicular diameters for each spot and using that number to find the area
(remember/find/google the formula).Using excel or another graphing program of your
choice, plot the calculated average area of the spots at each volume that you have
pipetted. There should be one data point for each volume (the average of the areas of the
4 spots at that volume). Be sure to include error bars representing one standard deviation,
label your axes, and title your plot. Attach this plot and your filter paper in your note
book.
Trouble shooting questions/homework
WRITE DOWN THE ANSWERS IN YOUR NOTE BOOK
Consider the following 5 problems, what causes them, how can they be avoided or fixed?
1
2
3
4
5
Pipettor leaks
Pipettor breaks
Liquid disappears in pipet, will not come out
Inconsistent volumes dispensed
Consistent wrong volume dispensed