Metrics & Measurements: Lab 1
BIO 135: Biotechnology and Human Affairs
NAME:__________________________
Metrics & Measurements:
Using micropipettes and determining concentrations
Part 1: Measurements
A pipette (also called a pipet or a pipettor) is a laboratory instrument used to transport a
measured volume of liquid. Pipettes are commonly used in chemistry and molecular biology
research as well as medical tests. Pipettes come in several designs for various purposes with
differing levels of accuracy and precision, from single piece glass pipettes to more complex
adjustable or electronic pipettes. A pipette works by creating a vacuum above the liquid-holding
chamber and selectively releasing this vacuum to draw up and dispense liquid. Pipettes that
dispense between 1 and 1000 μl are termed micropipettes.
Micropipettors: Directions for use.
Different pipetters are used for different volumes. Micropitters commonly come in four (4) sizes:
0.5-2 μl, 2-10 μl, 10-200 μl, and 200-1000 μl. The top of the pipettor will indicate the size. On
some pipetters it will show the range (i.e 10-200 μl), on some it will show the maximum volume
(i.e. the P 200 above measures between 10 and 200 μl). DO NOT set the volume of the pipettor
outside of this range – it will break the pipettor and these are VERY expensive little tools.
A pipette has two “stops”, which are places where the pipette plunger naturally wants to stop. The
1st stop is used to obtain (or pull up) your sample, the 2nd stop is to release (or expel) your
sample.
To obtain a sample: Depress the plunger to the 1st stop, put the pipette in the liquid you wish to
measure and slowly release the plunger. Now you have the desired volume of liquid in your
pipette. NOTE: If you do not release slowly, the proper volume will not be drawn up.
To expel a sample: Place your pipet tip into the container you wish to release your volume. Gently
touch the pipet tip against the container wall. Press down to the 1 st stop AND KEEP GOING to the
2nd stop. The liquid will be released as the plunger moves to the 1st stop and the last remaining bit
will be expelled as the plunger moves from the 1st to the 2nd stop.
To eject a tip: Depress the large gray button on the top of the micropipette.
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Pipetting rules:
Avoid breakage:
• Never drop a micropipette.
• Never rotate the volume adjuster either below or above the range of the instrument.
• Never allow the plunger to snap up when liquid is being drawn into the tip.
Avoid contamination:
• NEVER attempt to use the pipette without a disposable tip in place!!
• Do not rest the pipette on a dirty surface.
• A pipette tip that is used to extract one liquid must never be used to extract another!
• Never let your pipet tip touch another liquid or surface.
• Never immerse the barrel of a micropipette in a liquid above the tip.
Proper measurement:
• Never allow the liquid in a pipette to rise up to the cotton barrier. The barrier will soak up your
volume and lead to an inaccurate measurement.
• Never let the plunger ‘snap’ or move to the stop on its own. This will cause your volume to be
inaccurate.
• Use the 2nd stop ONLY to release your sample. If you move to the 2nd stop to obtain a sample
your measurement will be inaccurate.
1. How many pipettors are in your box? List their volume ranges.
2. What is the 1st stop for? What is the 2nd stop for?
3. Why is important NOT to immerse the barrel (i.e. the white part above the tip) of a micropipette
into a liquid?
4. What will happen if you dial the pipetter volume outside of its designated range?
5. What will happen if you let the plunger ‘snap’ back to its original position?
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To perfect your pipetting skills, especially with the small volumes used in many areas of biological
research, you will pipette water samples in 10 ul, 50 ul, 100 ul. It will be necessary to repeat the
pipetting several times in order to get the desired precision.
Part I: Setting the Micropipette
1. Check that you have the right sized micropipette. There are four sizes in the lab.
2. Dial the desired volume.
3. Push the end of the pipette into the proper size tip. The yellow tips are for the P-20's and P200's; the larger blue tips are for P-1000's. The white tips will be for the P-2.
Part II: How to Take Up Sample with a Micropipette (use safety goggles!)
1. Before picking up the micropipette, open the cap or lid of the tube from which you are taking
fluid. (Or, have your lab partner do this).
2. Hold the micropipette in one hand, almost vertical; hold the tube in your other hand. Both
should be at almost eye-level.
3. Depress the plunger of the micropipette to the 1st stop, and hold it in this position, then...
4. Dip the tip into the solution to be pipetted.
5. Draw fluid into the tip by slowly releasing the plunger. Watch the liquid enter the tip.
Part III: Expel a Sample From the Micropipette (use safety goggles!)
1. Open the cap or lid of the tube into which you are ejecting the fluid.
2. Hold the micropipette in one hand, almost vertical; hold the tube in your other hand. Both
should be at about eye-level.
3. Touch the micropipette tip to the inside wall of the tube into which you want to expel the
sample. This creates surface tension to help expel the fluid from the tip.
4. Slowly depress the plunger of the micropipette to the 2nd stop to expel the last bit of fluid.
5. Remove the pipette from the tube, keeping the plunger depressed to avoid sucking any liquid
back into the tip. When the tip is free of the tube, release your thumb pressure.
6. Always change tips for each new reagent you need to pipette. To eject a tip, depress the large
gray button on the top of the micropipette.
References
http://courses.brown.edu/Arthur_Landy-BI0047_F02/gschdadd8.pdf
http://www-class.unl.edu/bioc433/experiment1.htm
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Practicing with a P-1000 Micropipettes (use safety goggles!)
Water Weight Exercise: When biotechnology technicians calibrate pipettes, they use water to set
the calibration points. This is based on the fact that 1 ml of water weights exactly 1 gram (1 ml of
H20 = 1 gram). In the following exercise, you will pipette H20 and weigh it.
1. Set the micropipette to the desired volume of 500 μL (Table 1) or 1,000 μL (Table 2).
2. Place a “weigh boat” on the digital scale. Record its mass in the table below: Before
3. Attach a new blue disposable tip to the shaft of the pipette. Take up of 500 μL water.
4. DISPENSE SAMPLE into the “weigh boat”.
5. Slowly let the plunger return to the UP POSITION. Record the mass of the “weighing boat” in
the table below: After.
6. Repeat steps 2 to 6 four more times.
7. % Error = [(observed mass-expected mass) / expected mass] X 100
Data Table 1: Weighing 500 μL of H20.
Trial
Mass of “weigh
Mass of “weigh
#
boat” (grams)
boat” (grams)
Before
After
1
(After – Before)
observed
(grams)
expected
(grams)
%
error
0.5
2
0.5
3
0.5
4
0.5
5
0.5
What was your average % error?___________
Data Table 2: Weighing 1000 μL of H20.
Trial
Mass of “weigh
Mass of “weigh
#
boat” (grams)
boat” (grams)
Before
After
1
(After – Before)
observed
(grams)
expected
(grams)
1.0
2
1.0
3
1.0
4
1.0
5
1.0
What was your average % error?___________
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%
error
Part 2: Metrics
The metric system is a decimal system of weights and measures based on the meter as a unit of
length and the gram as a unit of mass and liter as a unit of volume. The abbreviations for meters,
grams, and liters are m, g, and l (sometimes capital letters are used). Metric units of
measurement are based on multiples of 10 where a prefix is used to denote some multiple (or
fraction) of the basic unit. The metric prefixes are listed below.
Prefix
giga
mega
kilo
centi
milli
micro
nano
pico
Symbol
G
M
k
c
m
μ
n
P
Factor
109
106
103
10-2
10-3
10-6
10-9
10-12
Numerically
1,000,000,000
1,000,000
1,000
0.01
0.001
0.000 001
0.000 000 001
0.000 000 000 001
Name
Billion
Million
Thousand
Hundredth
Thousandth
Millionth
Billionth
Trillionth
In this class, we will be regularly using small volumes of liquid usually measured in milliliters (mL)
or microliters (uL). Other common units of measurement in biotechnology include nanograms
(ng). The concentration of DNA is denoted as ng/ul. For example, the amount of DNA template
included in a PCR reaction could be 1ul of DNA that is at the concentration of 5 ng/ul.
Metric Conversion Exercise: Complete the metric chart below by indicating the metric equivalent
of the measurement provided.
Measurement
Equivalent
1000 ul
_______ml
1000 ml
_______L
1L
_______ul
2.222 ml
_______ul
0.5 ml
_______ul
200 ul
_______ml
5 ng/ul
_______ug/ul
5 ng/ul
_______ng/ml
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To convert from one concentration to another, a commonly used laboratory equation is
C1V1= C2V2
where C = concentration and V = volume
EXAMPLE: Let’s say that you have a tube of DNA where the concentration is 25 ng/ul. But you
need 5 ng/ul of DNA to do your PCR reaction. You would like to do several PCR reactions so you
decide to make 100ul of 5 ng/ul DNA. To determine how much DNA is need and volume of TE
buffer needed to dilute, perform the calculation.
C1V1= C2V2
where C1 = = 25 ng/ul
V1 = unknown variable
C2 = 5 ng/ul
V2 = 100ul
To solve, plug the variables into the equation
C1V1= C2V2
25 ng/ul * V1= 5 ng/ul * 100 ul
Rearrange
V1= 5 ng/ul * 100 ul
25 ng/ul
Cancel the units
V1= 5 * 100
25 ul
V1= 500
25 ul
= 20 ul DNA needed
Now you need to dilute it. Many dilutions occur in water, but DNA is more stable in a buffer
solution. We will dilute in TE Buffer solution. How much TE do we add?
V2 = Total Volume
V2 = V1+ Buffer Volume
V2 = V1+ V TE to add
V2 = 20ul DNA + 80 ul TE Buffer
So to dilute a DNA sample of 25ng/ul to 5ng/ul, you can take 20 ul of 25 ng/ul DNA and add it to
80 ul of TE. This will yield 100ul of 5ng/ul of DNA.
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Concentration Conversion Exercise: You are a lab technician for a company called DNA
Genealogies. This company collects DNA from individuals all over the county and types their
mtDNA and Y chromosome types. Your supervisor wants you to make DNA dilutions. She wants
100 ul of 5 ng/ul for 5 DNA samples. You are given the stock DNAs and these samples have
variable DNA concentrations (C1).
You know that you need 100 ul (V2) of 5 ng/ul (C2). What is the volume of DNA (V1) that you need
and how much TE buffer do you add to dilute it (VTE to add )?
C1
V 1(V DNA)
C2
V2
25 ng/ul
5 ng/ul
100 ul
47 ng/ul
5 ng/ul
100 ul
56 ng/ul
5 ng/ul
100 ul
205 ng/ul
5 ng/ul
100 ul
6 ng/ul
5 ng/ul
100 ul
V TE to add (ul)
1.
2.
3.
4.
5.
Next, you are asked to make a series of dilutions but all of the dilutions have different volumes
and concentrations. Calculate the DNA and buffer volumes needed to make the following
dilutions. Note: There is a tricky one (see last question).
C1
V 1(V DNA)
C2
V2
25 ng/ul
10 ng/ul
20 ul
5 ng/ul
1 ng/ul
100 ul
50 ng/ul
5 ng/ul
50 ul
5 ng/ul
25 ng/ul
1 ul
100 ng/ul
5 ng/ul
1 ml
1.
2.
3.
4.
5.
Which of the above conversions is impossible to perform? Why?
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V TE to add (ul)