CLASS SET
Measurement Lab
DO NOT WRITE ON OR REMOVE FROM LAB
Now that you are proficient with calculations and conversions, you are ready to
become familiar with some of the essential tools of the biotechnician: the
micropipetter, the balance, and the centrifuge. These three tools are used every
day in bioscience labs around the world.
Always follow Good Laboratory Practices (GLPs) for every lab.
*Keep your area clear and free from any safety hazards on or around the lab bench.
*Perform only one experiment at a time.
*Gather all materials before you start.
*Set up a disposal area before your start.
*Label every container as necessary.
*Once a solution is drawn into a micropipetter, be ready to elute (empty ) it
immediately.
Materials:
Each group needs:
100 ml sugar solution (dyed any color but blue)
100 ml of clear water
100 ml of water dyed blue
small beakers or containers
1.5 ml microcentrifuge tubes (12 per group)
microcentrifuge tube rack
Micropipetters (range 2 - 1000 ul)
1-200 ul tips
100-1000 ul tips
permanent marker for labeling
gloves (optional)
Class Shares Materials:
Digital balance
Flask
Water
Weigh boats
Calcium Chloride soln
Potassium Phosphate tribasic soln
Microcentrifuge (DO NOT use without teacher supervision)
Procedure:
A. Organize your workspace
1.
Arrange everything you need so that it can be easily reached. (If you are
using sterile technique, you should wash the area with ethanol). Collect
everything (including paper towels) for your lab that isn't to be shared
among the class.
2.
Everyone in your group should practice measuring.
3.
You will be asked in the analysis to illustrate how you arranged and organized
your workspace, so take note of it at this time.
B.
1.
2.
3.
4.
5.
6.
Practicing with the Micropipetter
(GLP tip - NEVER lay a micropipetter down with a filled tip. The liquid
could ruin the mechanism)
Practice setting the micropipetters. Each person in your group should set at
least one and have it checked by other group members for accuracy. Look at
the top of the micropipetter to see what size it is. Remember that the
highest place value of the numbers on the top is the same as the place value
of the top number on the dial you set. Be sure to convert ml to ul and
show the calculations in the margin of your lab notebook.
Set out a graduated 1.5ml microcentrifuge tube in a rack ready to dispense
the liquid you measure in the next steps.
You will be pipetting 600ul of colored sugar solution. The color helps you see
how much you are measuring. Choose the correct size micropipetter and set
it to 600ul. While you are waiting to use the micropipetter, practice opening
and closing Eppendorf tubes with one hand (usually the non-dominant hand).
Place a tip on the end. Do NOT touch the tip. Leave it in the box and push
the micropipetter firmly into the end of the tip. The smaller tips fit both
the 2-20 and the 20-200ul micropipetters. The larger (blue) tips are for
the 100-1000ul micropipetters.
Using one hand, hold the micropipetter and press down on the plunger with
your thumb or index finger (whatever feels comfortable). Note that there
are 2 places the plunger stops. The FIRST stop is for filling and SECOND
stop is for delivering. Practice a few times until you can easily feel the
difference between the two stops.
Press down to the first stop. Place the tip only into the liquid. You may rest
the tip against the side of the container just under the water line to steady
it. If you put the tip in deeper liquid may drip from the outside when you
deliver it. Slowly release the plunger. If you release the plunger too quickly
the liquid may splash into the micropipetter and contaminate it. If you are
pipetting viscous (thick) liquids, such as the sugar solution you are using, it
won't be able to enter the tip fast enough and your measurement could be
7.
8.
9.
inaccurate. Be careful not to remove the tip from the liquid before it is
filled or you will get an air bubble in the tip and an inaccurate measurement.
If you released the plunger slowly and kept the tip in the liquid but you still
have a bubble, you probably pushed the plunger down to the second stop
instead of the first.
Without removing the tip from the beaker, dispense the liquid by pushing
the plunger slowly down to the first stop. Try not to make any bubbles.
Repeat step 6. Drawing up the liquid twice ( this is called "pipetting up and
down") can improve the accuracy of the measurement.
Dispense the liquid into 1.5ml microcentrifuge tube and be sure it is near the
0.6 mark (600ul = 0.6ml). This is just a check to make sure you used the
correct micropipetter and set it correctly.
Discard the tip in the appropriate container by depressing the ejection
button.
C. Mixtures and Labeling (each group does all measurements)
1.
You are going to measure different colors and amounts into 10
microcentrifuge tubes. Microcentrifuge tubes are often called "Eppendorf"
tubes. Eppendorf is a popular brand of labware. Using one glove to avoid
contamination, choose 10 microcentrifuge tubes, close them, and label the
lids 1-10. Always label on the top so that it can be read without removing
the tube from the rack.
2.
Mark the hinge of tube number one and on the side of this tube write a
notebook reference to show where you are going to record what each tube in
this series contains. The reference should be written in the margin as
[notebook no.]:[page number]. Since each of you has only one notebook, the
first number should be 1. The second depends on how many pages you have
used so far in your notebook. For example 1:9 shows that the contents are
recorded on page 9 of notebook number 1. This is a common method of
recording in labs.
3.
Open all of the lids of the microcentrifuge tubes so they are ready to
receive the solutions. Before you begin measuring, think of what will be the
most efficient way of dispensing the amount. If several of the tubes
contain the same liquid you can measure them all out before you change tips,
so long as you are sure you didn't touch the tip to the inside of a tube
containing some other solution. You may also want to first fill all of the
tubes that have the same measure of liquid so you don't have to change the
setting too often. Sometimes it matters which ingredient is put in first.
Always check (think) before you start if you are unsure.
4.
Measure the following amounts into the indicated tubes. Mix the contents
by "pipetting up and down" several times. DO NOT pipette so vigorously that
you make bubbles. This can degrade some sensitive solutions such as
enzymes. You may want to close the tubes as they are filled or move them
back one row to avoid accidentally filling the same one twice.
Micropipetter Practice
Tube #
Tube #
1
5ul blue
7
20ul blue
2
10ul blue
100ul clear
3
100ul blue
8
20ul blue
4
1000ul blue
500ul clear
5
5ul blue
9
200ul blue
20ul clear
800ul clear
6
20ul blue
10
500ul blue
20ul clear
500ul clear
Check your accuracy by setting the micropipetter to the total amount in the
microcentrifuge tube and drawing all of the solution into the tip. Your pipetting
was accurate if you leave no solution behind and have no air bubble in your tip. The
amounts in tubes 1 and 2 are so small that if any is clinging to the side you won't be
able to draw it up. If this is the case, you may use the microcentrifuge (tell your
teacher first). Place your tube and the "balance tube" in the microcentrifuge for
10 seconds to "spin down " the liquid so it all collects in the bottom of the tube.
Class Shares Station(s)
D.
1.
2.
3.
4.
Measuring volumes with the balance
You will be using a digital scale (pan balance) for most of your lab work.
These balances are generally used for weighing amounts larger than 0.05g
and are typically accurate only to 0.01g.
Make a sketch of the balance used in the lab in the margin of your notebook.
Label the on/off switch and the tare button. Also include the balance
identification/serial number. Most GLPs require that this be listed for
every weight measurement.
Turn on the balance. Place a weigh boat on it and press "tare". It should
reflect 0.0 grams.
Now , using the correct seriological pipette, dispense the first amount of
water into the boat. Record the weight in grams. Continue adding to each
amount (without emptying the weigh boat), recording the total weight each
time.
1.
2.
3.
4.
5.
Press A and squeeze bulb simultaneously
Place tip of pipette in solution and press S to
suck up desired amount
Transfer pipette tip to new container
Press E to elute (empty desired amount of
liquid)
By pressing S and the bulb simultaneously the
last amount of liquid can be released.
 DO NOT GET COTTON PLUG WET
Weights of Water ( in grams)
Procedure
Weight
0.0 g.
After taring
After 1st 5.0ml
After 2nd 5.0ml
After 3rd 5.0ml
After 4th 5.0ml
* 1 g. = weight of 1000 ul.
E.
Accuracy of glassware in measuring
1.
Measure out 10ml of water using an Erlenmeyer flask or beaker. Tare a
weigh boat on the balance. Pour the water into the weigh boat. Record the
weight. Pour the water back into the flask.
2.
Measure out 10ml of water using a graduated cylinder. Pour the water into
the tared empty boat. Record the weight. Pour the water back into the
flask.
Accuracy of Glassware ( in grams )
Glassware used
Weight of water (g.)
Erlenmeyer flask
Graduated cylinder
F.
1.
2.
3.
Using a Microcentrifuge
Label microcentrifuge tube CaCl2 + K2PO3.
Add 500ul Calcium Chloride and 500ul Potassium Phosphate
*Make certain that you change the tip first. DO NOT crosscontaminate.
Place in microcentrifuge. Each tube should have a tube of equal weight
across from it for balance. Caution: An out-of-balance centrifuge is
hazardous.
4.
Spin for 1 minute. You are looking for a precipitate to form. The liquid above
the precipitate is called the supernatant.
Dry measures:
Measure each of the items set out and record in a table entitled “Dry
Measurement”.
Once you have completed ALL activities, you may begin the analysis
portion of the lab. Lab books are to be turned in at the FRONT of the
room. Replacement of the lab book on the side table, on or after the
due date, will ensure that it is NOT graded on time; constituting a late
grade.
You MUST copy the analysis/questions beginning at the top of the next
available page. Make sure you write your answers immediately following
each question. Failure to follow proper procedure will result in point
deductions.
Measurement Lab Analysis
This should begin on at the top of a separate page following your pre-lab write-up.
Copy each question and leave room directly below the question for your answer.
Part A:
1.
Make a sketch to show how you organized your lab space.
Part B:
0
2.
If a 20 - 200ul micropipetter is set to :
4
How many ul is it set to measure?
3
How many ml does this equal ?
3.
Why should you avoid touching the micropipetter tips when you are putting
them on?
4.
Why should you avoid putting the micropipetter tip too deep into the liquid?
5.
What happens when you push the plunger to the second stop before drawing
up the liquid?
6.
What does the phrase "pipetting up and down" refer to?
Part C:
7.
Where should microcentrifuge tubes be labeled?
8.
What type of caution should you use when using a centrifuge ?
9.
What does the reference 24:127 refer to on the label of a microcentrifuge
tube?
10.
Describe the order in which you filled the tubes in step 4 for maximum
efficiency .