Introduction [PIPETTE WARS: QUALITIATIVE AND QUANTITATIVE

MICROPIPETTE SKILLS]

Before Coming to Lab: Overview

1. Read the entire lab entitled “Pipette Wars” (first In the laboratory of Cell Biology, you
part of this worksheet) will frequently use micropipettes to
transfer small volumes of liquids.
2. Watch this instructive video (9 minutes) on You will also regularly create and
using a micropipette: use standard curves to analyze and
https://www.youtube.com/watch?v=uEy_NGDfo_8 assess the data you have collected.
Both techniques are commonly
encountered in the real world of
science. This exercise is designed to
familiarize you with them. You will
use micropipettes to construct a
standard curve.

Perform this exercise in pairs

Objectives of this Laboratory Exercise

At the end of this lab students will be able to:

• Know how a spectrophotometer works.
• Use micropipettes to create a standard curve.
• Use the standard curve to determine the concentration of an unknown protein
solution.

Pipettes versus Micropipettes

Pipettes are slender, graduated tubes, usually of glass or

clear plastic, used to measure the volume of fluids or to
transfer them between vessels. Micropipettes, sometimes
called pipettemen, are mechanical devices used to
accurately measure small volumes of liquids, usually less
than 1 milliliter (mL). There are two basic types of
micropipettes, fixed volume, and adjustable volume. As
the name implies, fixed volume micropipettes are preset at
the factory to deliver a single volume every time they are
 used. Adjustable volume micropipettes, like the ones we
use in this Laboratory, can be set to deliver a fairly wide
range of volumes. These are manufactured to deliver
different ranges of volume, e.g. one might deliver volumes
in the range between 1 and 100 L and another volume in
the range between 100 and 1000 L.

The ability to accurately measure and transfer small

volumes of liquid is essential to your success in this
Laboratory. In use, the micropipettes itself should never
touch the liquid that is being transferred. Instead, a
disposable tip is placed on the end of the device and the
tip is submerged in the liquid. Never use a micropipette
without a tip. To avoid cross-contamination between
liquids, change the tip between each use. When in doubt,
change the tip. Tips are color-coded. In general, blue
pipette tips are used with micropipettes with capacities of
200-1000 µl, yellow tips are used with micropipettes with
capacities of 5-200 µl, and clear (“natural”) tips are used
with the smallest micropipettes, those with capacities of
0.5 to 10 L. Table 1 defines some units of volume that
you will encounter in this course and in the scientific
literature.

Table 1. Units of volume

Unit Abbreviation Definition
Liter L
Milliliter mL 1/1000th of a liter (equal to a cubic centimeter or
“cc”)
Microliter L 1/1000th of a mL
Nanoliter nL 1/1000th of a µL
Picoliter pL 1/1000th of a nL
How to operate a micropipette

General considerations
• Handle these devices with care. Rough handling can
cause them to lose calibration, and, therefore,
accuracy. Dropping them can cause irreparable
damage.
• Never force an adjustment knob. If the knob becomes
hard to turn, it is probably at the limit of its travel.
Forcing it can cause irreparable damage.
• Never use a micropipette outside of its specified
range.
• Do not use micropipettes to measure strong acids,
bases, or flammables.
• For the greatest accuracy in volume measurement,
select the smallest size pipette that will handle the
volume you wish to transfer. Accuracy is reduced
when an unnecessarily large pipette is used to
measure small volumes.

General operation
1. Select the most appropriate sized micropipette for the
job at hand.
2. Set the desired volume by turning the adjustment
knob as shown in Figure 1 (Micropipettes from
different manufacturers have different adjustment
mechanisms. Check with your instructor if the
 micropipette you are using does not look like the one
in Figure 1.)
3. Put on the appropriate sized tip by tapping the
micropipette into the tip while the tip is still in its rack.
Do not touch the tip.

Figure 1. Adjusting the desired volume on a micropipette.

4. The push lever of the micropipette has 2 “stops," or
depression lengths, where you will feel resistance to
the pressure of your thumb. Figure 2 shows the
proper operation of a micropipette. Before putting the
tip into the sample solution, depress the thumb knob
to the first stop.
5. Immerse the tip approximately 3 mm into the sample
solution (step 1).
6. Slowly release the thumb knob to the initial position
(step 2). Do not release it too quickly, the sample
might cavitate and form a bubble, destroying the
accuracy of your volume measurement.
7. Withdraw the tip from the sample solution.
8. Place the tip against the sidewall of the receiving
container (step 3).
9. Smoothly depress the thumb knob to the first stop
(step 4), pause, then depress the knob to the second
stop (step 5).
10. Remove the tip from the receiving container and
return knob to the initial position. Do not let the knob
snap back.
 11. Remove the disposable tip into the indicated waste
container by firmly depressing the tip ejector knob
(step 6).
12. Add a new tip and continue.

Figure 2. Operating the micropipette

Lab
procedure [PIPETTE WARS: QUALITIATIVE AND QUANTITATIVE
MICROPIPETTE SKILLS]

The lab procedure will be explained by your TA in a

recording and a presentation. Fill the information required
with the data provided by your Professor or TA.
ACTIVITY 1: USING THE SPECTROPHOTOMETER
The purpose of this activity is for you to learn how the
spectrophotometer works and why it is a common tool
to measure colorimetric reactions.
First, we will take a few minutes to review how the
spectrophotometer works. This instrument will be our main
measurement tool for many labs this semester. The
spectrophotometer is used to measure the concentration
of compounds or particles in a solution based on the
amount of light that passes through the solution.
Part A: What is inside a spectrophotometer?
1. Light source - This is a lamp that produces white light
(a mixture of visible wavelengths).
2. Slit - This controls the amount of light being passed
through the sample.
3. Wavelength selector - The white light is split into its
component wavelengths by a prism, and
monochromatic light (a single wavelength) is then
selected by rotating the prism so that the desired
wavelength of light passes through the sample.
4. Sample holder - This keeps the sample you are
measuring in the proper position.
5. Light detector - This is a photosensitive cell that
measures the light that gets through the sample.
6. Meter - This gives the readout from the photocell
(Transmission/Absorbance)

Schematic diagram of Spectrophotometer

(Carolina.org)

Part B: Observing different wavelengths:

Turn on your spectrophotometer (Spectronic 200E) and
 allow it to warm up (about 5 minutes). You can leave it on
for the rest of the lab period. Read the Spec User Guide.
With the wavelength set at 650nm, BLANK the Spec with
a cuvette filled with water. Then place a sample tube of
colored water into the sample chamber and adjust the
wavelength down each 20 nm to 450nm. In the table
below, note the maximum wavelength that gives an
absorbance reading. Then indicate what color
corresponds to the light absorbed by the tube of food
coloring.
Color of solution Wavelength (nm) when light is Color spectrum that is absorbed
absorbed
Red 553 green
Blue 602 Orange
Green 610
Yellow 472
orange
blue
-

ACTIVITY 2: CONSTRUCTION OF A STANDARD CURVE

Part A: Measuring absorbance accurately with a
spectrophotometer:
In order to use the spectrophotometer in Activity 2 to
measure the concentration of a substance (red food
coloring, in this case), you will take advantage of the fact
that the amount of light absorbed is linearly proportional to
the concentration of the substance. Therefore, you will be
concerned with something called "Optical Density" (OD),
which is a measure of how effective the substance is at
blocking (being dense to) light. High OD corresponds to
strong absorbance, and strong absorbance is the
consequence of high concentration.
In order to make accurate measurements with a
spectrophotometer, there are two main factors to consider:
1. You must set the limits of possible measurements:
a. One limit corresponds to infinite absorbance. That
is, none of the light is transmitted through the sample.
You will use a mechanical shutter inside the
 spectrophotometer to block all the light, and thus
simulate infinite absorbance. This is done with the "0
Set" button ("0" in this case corresponds to 0%
transmittance of light). With the "0 Set" button
depressed, set the absorbance to infinity (%
transmittance = 0).
b. The other limit corresponds to no absorbance.
That is, all the light is transmitted through the sample.
You will use a solution that contains everything except
the substance you want to measure (a so-called
"blank") to set the baseline absorbance to 0. Your
blank should contain 3 ml of buffer solution and one
aliquot of an enzyme. Because there is no substrate
present, no product will be produced. By setting
absorbance to 0 for the blank, only the newly
produced material will be sensed by the machine
when you do a real experiment with a substrate
present.
2. You must minimize extraneous influences on light
readings.
To do this, you should take the following precautions:
always place the cuvette in the Spec in the same
orientation, wipe the tubes before placing them in the
Spec, and always close the lid of the sample chamber
when taking a reading. These steps will reduce
variation due to the unevenness of the glass or dirt on
the tubes.
An assay is a test or an evaluation. The term often refers
to an experimental procedure used to estimate the amount
or concentration of a substance in a sample. Such assays
are often based on the measurement of a physical or
chemical property of one compound that changes in the
presence of the compound of interest. The measured
property might be fluorescence, the ability to form a
precipitate, optical density, radioactivity, or, in today’s
case, a color change.

A standard curve is a graph of known amounts of an

analyte (X-axis) versus the absorbance of each known
concentration (Y-axis). Standard curves can be linear or
curved. Once a standard curve has been created, it can
be used to determine the amount or concentration of the
substance of interest in other samples (unknowns).

In Activity 2, we will graph increasing parts per million

(ppm) of red food coloring versus the absorbance
reading of each known concentration. Based on your
work with the spectrophotometer above, you should select
an appropriate wavelength to read the absorbance of red
solutions. Importantly, you will test your skill in using the
micropipettes by constructing a standard curve for ppm of
red food coloring versus absorbance. If you use the
pipettes correctly, the curve that you generate will be a
straight line with an R value of 1.00. R is an estimate of
2 2

the “goodness of fit” of the graphed line, called the

trendline, to the behavior of the data points you acquired.
Values of R range from 1 (perfect linear fit) to 0 (no fit).
2

Part B: Procedure

1. Read the Spectronic 200E User Guide

2. Set the appropriate wavelength for this activity.
(Hint: Wavelength of absorption of red light)
3. Collect 6 large test tubes for making dilutions and se
them in a rack. Label tubes 1-5 and “unknown” with a
marker.
4. Using the 100ppm solution of red food coloring
provided, prepare 5mL volumes of 5ppm, 20ppm,
C(UI = C2V2

x1 = 100
pm.
Vi =
?
22
=
5, 20, 40, 60, 80. absorbance.
v2 5 ml water
reddye
=

I
100(v) =
(5)(5)
V1 =
25/100 = 0.25 all 4.75 O
5

(100)(V) (5)20) =

Y 0.02
-

0
a
"of
=

0.04

v4 (())yy
=
=
2
3

0.06

too be
the
"so
-

~
80 =
() (00) =
4N. I 0.08.
500
#

equation from graph:

=
y= 0.001n
-0.003, R =
0.9972.

0.04 + 0.003 =
0.001

0.04 3 = x

0.001

Usppon concentration of unknown.

 40ppm, 60ppm, and 80ppm by diluting the stocks in
the appropriate volume of water.
5. Measure Absorbance.
a. Blank the spec. Fill the cuvette with dH2O (blank
solution) and wipe away fingerprints with
Kimwipe. Place the cuvette in the spec and
press “0.00”
b. Fill and read the absorbance of each red food
coloring concentration.

Data collection table for red food coloring standard

curve:

Tube # ppm Absorbance

Replicate 1
BLANK 658
1 44H
2
3
4
5
UNKNOWN

Now you will use the Standard Curve of red food

coloring ppm versus absorbance to determine the
concentration of an unknown sample.

1. Transfer 3ml of an unknown into the cuvette and read

the absorbance three times (in triplicate).
 2. Now that you have the absorbance of this unknown,
you can utilize the relationship you derived between
absorbance and ppm in the Standard Curve trendline
to determine the ppm in your unknown.

2
Worksheet [PIPETTE WARS: QUALITIATIVE AND QUANTITATIVE
MICROPIPETTE SKILLS]

Before you start:

• Read the entire lab exercise
• View the entire Micropipette Tutorial.
• Answer the questions below
PART A. Micropipettes

1. Select the appropriate pipette to achieve the following

volumes.

Pipette options Target Volume Your selection

• P1000 15 L
• P200 u
• P20
• 10 ml
• 5 ml
• 1 ml

970 L
N
7 ml
2 ml
200 L
80
520 Li L

2. Create a numbered list of steps to operate a

micropipette and eject 185 L of a solution into a
microcentrifuge tube. Begin with your pipette selection
and end with ejecting the pipette tips into a waste
container.
3. If you press down a micropipette to the 2 stop, insert
nd

the pipette tip into solution, and then raise your thumb, will
you aspirate the target volume? circle YES or NO.

If no, will the volume be HIGHER or LOWER than the

target volume? (circle the correct choice)

4. In the left panel, fill in the correct numbers in the

corresponding “micropipette window.”
In the right panel, write the micropipette and volume that
corresponds to the numbers in the windows.
 I O I

957

7 9 8

lou
10

OM1 's5%us 6.5 l

5. A) Describe how to use a 100ppm dilution to achieve

25mL of a 10ppm dilution.
V2 22

C2U2
(,V1
=

(100)(x) (25)(10)
=

-u 2.5M.
(2)(y)E
=

2 x
=

Worksheet [PIPETTE WARS: QUALITIATIVE AND QUANTITATIVE

MICROPIPETTE SKILLS]

PART B. Standard curve

Using the Excel software, plot the “standard curve,” or

absorbance versus ppm. Which values should be
plotted on each axis?
absorbance
Y-axis: _____________ ppo
X-axis: _______________

a. Should this line be straight or curved, or both?

straight
_________________________

b. If your points begin to plateau at high ppm

concentrations, what can you infer about the
sensitivity of the spectrophotometer to light at
high ppm concentrations?
sensitivity lowers.
_______________________
the
c. Should you incorporate the “saturation points”
into the linear trendline for the ppm standard
curve? NO

d. What is the general formula for any line?

________________
y
mx
= b +

e. What is the R value for your trendline?

2

______________________
R== 997.2

R -values are a measure of how well

2

the data points form a linear line, aka

the “goodness of fit” of your data.
Your R -value should be 0.93 or
2

higher. It is critical that you have

good pipetting skills to work
productively.

f. Why is your pipetting accuracy when transferring

small volumes to tubes 1-5 important to achieve
a good R -value?
2

_______________________________________
if we don't pipette right,
the dilutions will

_______________________________________
be
wrong.
So
graph will be wrong.
_______________________________________
___________

g. What does the R value of your standard curve

2

tell you about your pipetting skills?

_______________________________________
good pipetting skills.
___________________
 Paste your plot here

h. Think about how you can utilize the standard

Curve to determine the ppm in the unknown.
Describe your strategy here:

i. What is the ppm of your unknown?

__________________