BIO206 Laboratory 1

Spectrophotometric analysis of DNA and proteins

Before Lab:
• Carefully review the safety guidelines from the Pre-lab exercises. You will not be
permitted to participate in labs without appropriate safety clothing.
• Review dilutions and serial dilutions from the Pre-lab exercises.
• Read and understand all lab exercises and their background theory. There are Pre-lab
Quizzes for all Labs including this one.

After Lab:
• Hand in Laboratory 1 Worksheet to Quercus before 10 pm one day after the day of your
lab

Lab 1 outline
Accurately quantifying concentrations of DNA and proteins in solution is a basic exercise in
molecular biology labs. During these experiments, you must handle small volumes of reagents
using micropipettes to do dilutions and serial dilutions.

Lab 1 is a three-part lab. Part A will train you how to use micropipettes. In Part B, you will use
measure the concentration of an unknown DNA solution using Salmon sperm DNA as an example.
Part C is a read-only lab which reviews spectrophotometry of protein solutions.
• Part A: Introduction to Micropipettes
• Part B: Quantification of DNA solution using spectrophotometry
• Part C: Quantification of protein solution using spectrophotometry (Read-only, still
testable material in Quizzes etc.)

Safety guidelines for Lab 1

Salmon sperm DNA is typically harmless to human body, but sill handle them with caution. Rinse
with running water immediately and thoroughly if it gets on your skin.

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Part A1: Introduction to Micropipettes / Practice exercise
In a molecular biology lab, you routinely handle volumes of reagents in the microliter (μL) range.
One microliter is 1/1000 of 1 milliliter (mL, 1 mL = 1000 μL), a volume too small to be measured
using a graduated cylinder. Micropipettes are used to handle these small volumes with high
accuracy (see Appendix A for metric conversions).

Micropipettes comes in different sizes

Micropipettes comes in different sizes, each crafted to handle a specific range of volumes (Figure
1). For example, a P-10 micropipette handles volumes between 0.5 μL- 10 μL whereas a P-200
handles 20 μL- 200 μL. Every micropipette MUST be used together with the corresponding
disposable tips. Handling solutions without these tips will contaminate/damage the micropipette.

The volume range for some micropipettes overlap, such as the P-10 (0.5 μL- 10 μL) and the P-20
(2 μL- 20 μL). When pipetting volumes within this overlapping range, use the smallest micropipette
which can handle the volume in one go. For example, use a P-10 to pipette 5 μL, and not P-20.

Figure 1. Micropipettes. a) Two "1000 μL" pipettes from different manufacturers with major
features labelled. The volume indicators are on their sides and can not bee seen here. For some
pipettes (such as the Rainins), the plunger also acts as the volume adjustment knob. b) Different
sizes of Rainin pipettes shown with their corresponding tips. Traditionally, pipettes for ~10 μL,
~100 μL and ~1000 μL are colour coded red, green and blue, respectively.

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Volume indicators
The volume indicator shows the volume of solution which the micropipettor is set to handle
(Figure 1a). Reading these may be confusing at first since the digits of volume indicators are
different for differently sized micropipettes. Use Figure 2 as a reference while learning how to read
volume indicators. You don't need to memorize all of this at once. With some experience, this
becomes second nature.

The volumes of these micropipettes can be adjusted by simply turning their volume adjustment
knobs. Some micropipettes such as the Rainins have a locking mechanism which needs to be
disengaged before adjusting their volumes (the 'locked' and 'unlocked' positions beside the
plunger can be seen in Figure 1b). Don't forget to lock it again before use.

All volume-adjustable micropipettes have a minimum and a maximum volume which they can
handle. NEVER adjust their volume knobs past this range as this may cause damage.

Figure 2. Examples of volume indicators for typical micropipettes. Minimum and maximum
volumes for each micropipette is shown alongside examples in between.

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'First stop' and the 'Second stop' of the micropipette plunger
After setting the volume and putting on the pipette tip, hold the micropipette upright and press
on the plunger with your thumb to uptake/dispense liquids. The plunger is operated in a two-step
mechanism, corresponding to the two positions which the plunger stops moving while you gently
press on it.

Practice operating the plunger in mid-air before you handle liquids.

1. Get a P-1000 micropipette and put on the appropriate tip.
2. Set the P-1000 to 500 μL and hold it upright.
3. Using your thumb, press on the plunger gently. Eventually, you should feel a firm resistance
and the plunger stops moving so long as you are only applying gentle force. This is the first
stop.
4. Then, push harder on the plunger. The plunger should move past the first stop and goes
all the way down. This is the second stop.

Micropipette practice exercise

Make sure you have practiced using the plunger before you proceed.

Materials
• Coloured water
• 1.5 mL microcentrifuge tubes

Procedure (done individually)

1. Obtain a new 1.5 mL microcentrifuge tube.
2. Follow the step-by-step instructions in the 'Using a micropipette' section (on next page:
page 5) to pipette 500 μL of coloured water into the 1.5 mL tube.
3. IMPORTANT: Self-check if you have accurately pipetted 500 μL into the 1.5 mL tube using
the graduation marks on the tube. You must confirm with your TA that you know how to
use a pipette correctly before proceeding.
4. Once you are comfortable with the P-1000, practice pipetting 150 μL, 75 μL, 15 μL, 5 μL
and 2 μL into the 1.5 mL tube using appropriate micropipettes.
• Pipette these volumes into the same 1.5 mL tube. Feel free to get a new tube if
necessary.

Cleaning up
• Throw away the 1.5 mL tube with its contents into regular waste.
• Leave the coloured water on bench.

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Using a micropipette
• Micropipettes are fragile and expensive to replace. Please use with care and respect.
• Every micropipette has a minimum/maximum volume it can handle. NEVER force the
volume knobs past limits.
• Always use micropipettes with a tip.
• Always hold micropipettes upright; don't hold them upside down.

1. Chose the appropriately sized micropipette for the volume you are handling.
2. Adjust the volume to desired amount. Make sure you unlock/lock the micropipette
appropriately if your micropipette has a locking mechanism.
3. Obtain a box of the corresponding tip and firmly press the micropipette into one of the
tips.
• Do not 'bang' on the tips while putting them on. This is unnecessary.
4. While the tip is in mid air, press the plunger to the first stop. Hold the plunger at the first
stop, and while holding, immerse the tip into the solution (Figure 3).
5. Slowly release the plunger to its home position. Observe the tip as the solution gets drawn
in. Keep the tip immersed in solution until the plunger returns to the home position
completely.
• Releasing the plunger too quickly results in inaccurate pipetting such as air bubbles
getting introduced into the pipette tip.
6. Withdraw the tip from the solution.
7. Aim the end of the tip into the container which you want to dispense the solution.
• If possible, place the end of the tip gently against the wall of the container. This
makes the solution 'stick' to the container, making it easier to dispense everything.
8. Gently press the plunger to the first stop, which will dispense most of the solution. Then,
press the plunger to the second stop to completely dispense the rest of the solution
remaining in the tip. Hold the plunger at the second stop and pull the tip out of the
container.
9. Return the plunger to the home position.
10. You can keep using the same pipette tip so long as you are pipetting the same solution into
a clean container. Otherwise, eject the tip into the waste container using the tip ejector.

Figure 3. Inserting pipette tips into solution. Try to hold the end of the pipette tip close to the
surface of the solution. Avoid inserting the tip deeply into solution as this may cause some of the
solution to stick on the surface of the tip.

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Part A2: 'Reagent mixing' exercise
The 1.5 mL microcentrifuge tube is commonly used to set up reactions in a molecular biology lab.
The following exercise simulates setting up reactions in a 1.5 mL tube. When setting up these
reactions, add the reagent with the largest volume first to the tube since it's usually easier to
handle larger volumes of solution. Rest of the reagents can be added directly into the first solution.

Reagents in 1.5 mL tubes can be mixed by gently flicking the tube with your fingers and/or using a
vortex (Appendix B). After mixing, it is a good practice to centrifuge the tubes briefly using a
microcentrifuge (Appendix C) to collect all solution to the bottom of the tube. You don't want
drops of reagents remaining on the side of the tubes, not participating in the reaction.

A scientific equipment is only as accurate as their users. You can't assume that you will pipette
accurately just because you are using a well calibrated micropipette. Pay close attention and
handle the equipment appropriately to bring out their maximum performance.

Materials
• Solution I
• Solution II
• Solution III
• Solution IV
• 1.5 mL microcentrifuge tubes

Procedure (done individually)

1. Obtain three, clean 1.5 mL microcentrifuge tubes and label each A, B, and C using a
permanent marker.
• Label 1.5 mL tubes on their lids. It's easier to see the labels this way.
2. Add reagents to Tube A following Table 1.
• Make sure to use fresh tips for different reagents.
• Begin by adding the largest volume first.
3. After pipetting all reagents into Tube A, mix well by vortexing / flicking with your fingers.
4. Put Tube A in a tabletop mini-centrifuge in a BALANCED way and perform a short spin
(Appendix C). This pulls down all liquids to the bottom of the tube.
• Balance tubes with your group members.
5. When made correctly, Tube A should contain 200 μL of solution. Double check this by
setting a P-200 to 200 μL and pipetting the contents of Tube A. If Tube A contained exactly
200 μL, the pipette tip should fill without air bubbles at the end while leaving any solution
in Tube A.
• Other results indicate pipetting error. Have your TA check your pipetting technique.
6. Repeat the exercise for Tubes B and C and double check pipette accuracy.
• Double checking pipetting accuracy using a micropipette is not always necessary.
However, using a micropipette to 'measure' volumes of solutions is a fairly common
trick used in labs.

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Table 1. Reagents to add to Tubes A, B and C.

Volumes to add (μL) Total volume (μL)

Solution 1 2 3 4
Tube A 40 12 70 78 200
Tube B 13 30 7 0 50
Tube C 3 5 0 2 10

Cleaning up
• Throw away the 1.5 mL tube with its contents into regular waste.
• Leave reagents I - IV on bench.

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Part B: Spectrophotometric Analysis of Nucleic Acids
Absorbance spectrophotometry is commonly used to measure the quantity and quality of nucleic
acids in solution (read Appendix D for more on spectrophotometry). For nucleic acids, the amount
of ultraviolet (UV) radiation, set at 260 nm wavelength, absorbed by your sample is used to
calculate its concentration (absorbance at 260 nm = optical density at 260 nm = OD260). The
relationship between OD260 and sample concentration varies for different types of nucleic acids.
For double-stranded DNA (dsDNA), OD260 of 1 corresponds to 50 µg/mL, assuming that the
solution was measured using a cuvette with a 1 cm path length. For single-stranded DNA (ssDNA)
or RNA, OD260 of 1 corresponds to 40 µg/mL.

Purity of samples can be assessed using absorbance measurements at other wavelengths. The
ratio of OD260 and OD280 is commonly used for this purpose (OD260/OD280). Pure solutions of DNA
or RNA have OD260/OD280 of 1.8 and 2.0, respectively. OD260/OD280 smaller than these values
typically indicate contamination by proteins and/or aromatic compounds such as phenol; the
bigger the difference is between the measured ratio and the theoretical value, the more
contaminated the solution. In addition, absorbance at 230 nm (OD230) can be used to indicate
contamination by urea as well as aromatic compounds.

As an example, let's say you measured a dsDNA solution which gave the following results:
• OD260 = 0.70
• OD280 = 0.39

Remember that for a dsDNA solution:

• OD260 of 1 = 50 µg/mL
• OD260/OD280 of 1.8 = pure solution of dsDNA

Therefore, for your dsDNA solution:

• Sample concentration = (50 µg/mL)(0.70) = 35 µg/mL
• Sample purity = 0.70/0.39 = 1.79 = this is a very pure (but not perfect) solution of dsDNA,
as 1.79 is very close to the theoretical value for dsDNA, 1.8

In the following exercise, you will analyze a DNA solution of unknown concentration using
spectrophotometry.
• Note 1: You suspect that the concentration of this solution is too high to be measured
directly, and therefore decide to serially dilute the original solution before analysis. You
are diluting the original solution to a final dilution factor of 200x (Figure 4).
• Note 2: We are using the Nanodrop spectrophotometer to analyze these DNA solutions
(Figure 5). The Nanodrop allows us to analyze samples using a much smaller volume
compared to a regular spectrophotometer. It also automatically calculates the
concentration/purity of your DNA solution, but you are still required to be able to do these
calculations manually.

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Figure 4. Schematic diagram for diluting salmon sperm DNA. Take note that the third dilution step
is a 2x dilution, not 10x.

Figure 5. Small-volume spectrophotometers. a) Thermo Scientific NanoDrop 8000

spectrophotometer. b) Mettler Toledo UV5 Nano spectrophotometer.

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Reagent handling techniques recap
• Use fresh tips when pipetting a different reagent.
• Mix reagents well after combining different reagents together.
• Once done mixing reagents, centrifuge the 1.5 mL microcentrifuge tubes to collect its
contents at the bottom of tube.

Materials
• Salmon sperm DNA solution (concentration unknown)
• deionized water
• 1.5 mL microcentrifuge tubes
• Nanodrop spectrophotometer (or equivalent)

Procedure (done individually)

1. Obtain a clean 1.5 mL microcentrifuge tube and label it 'H2O'. Transfer 1.0 mL of deionized
water to this tube. Use this aliquot of water to set up your dilutions.
• Using smaller aliquots of reagents (in this case, water) to set up reactions reduces
the numbers of times you open the larger storage container, lowering chance of
contamination.
2. Plan your serial dilution for the original DNA solution by completing Table 2. A schematic
of this dilution is shown in Figure 4. The total volume at each step of dilution should be 100
µL, and the final concentration relative to the original should be 200x.
• Show your calculation to your TA before you proceed.
3. Set up your tubes for serial dilution. Obtain three, clean 1.5 mL tubes and label them
appropriately for the three dilution steps. Then, pipette the required volume of your
diluent into each tube.
4. Start your serial dilution. Perform the first dilution by pipetting the required volume of your
original DNA solution into the first tube. Mix very well and centrifuge briefly (no more than
3 - 4 seconds, using the 'short' spin function; Appendix C) to collect all solutions to the
bottom of the tube by centrifugation.
5. Perform the second dilution by pipetting the required volume of your diluted DNA (in your
first tube) into the second tube. Mix and centrifuge.
6. Perform the third dilution by pipetting the required volume of your diluted DNA (in your
second tube) into the third tube. Mix and centrifuge. Your dilution is complete.
7. Analyze your third dilution using the Nanodrop spectrophotometer (or an equivalent
equipment). Your TA will assist you in this process.
8. To the Lab 1 Worksheet, record OD260, OD280 and the DNA concentration of your diluted
salmon sperm DNA solution. Remember, this is not the concentration of the original DNA
solution given to you.
9. Complete the Lab 1 Worksheet using your data.

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Table 2. Serial dilutions for salmon sperm DNA solution. The first dilution step is a 10x dilution of
the original DNA solution. The second dilution step is a 10x dilution of the first dilution. The third
dilution step is a 2x dilution of the second dilution.

Volume of DNA Volume of Total volume Dilution factor

sample to add diluent to add after mixing (μL) relative to
(μL) (μL) original solution

Tube 1 100 10x

(dilution step 1)

Tube 2 100 100x

(dilution step 2)

Tube 3 100 200x

(dilution step 3)

Cleaning up
• Throw away all tubes with its contents into regular waste (including original DNA solution,
dilutions, etc.).
• Keep deionized water on benchtop.

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Part C: Spectrophotometric Analysis of Proteins (Read-only)
Concentrations of proteins in solution can also be determined by spectroscopy. Here, we will use
the protein Bovine Serum Albumin (BSA) as an example.

UV light between 270 nm - 280 nm is absorbed by aromatic amino acids tyrosine, tryptophan and
phenylalanine. A simple way to determine concentration of a protein solution is to measure the
sample between 250 nm to 310 nm. This typically produces a bell-shaped absorbance curve with
the maximum absorbance (AbsMAX) occurring at around 275 nm (Figure 6). For BSA in solution
measured using a 1 cm path length cuvette, absorbance of 1 at AbsMAX = 1 mg/mL of BSA.

Figure 6. Spectrophotometric analysis of BSA using the BIO-RAD SmartSpec Plus Spectrophotometer.
BSA solution was measured between 250 nm - 310 nm wavelength. For this sample, AbsMAX occurred at
272 nm with absorbance of 0.129 Absorbance Units. BSA concentration = (0.129)(1 mg/mL) = 0.129 mg/mL

Different proteins absorb UV light with different rates as each protein has different amino acid
compositions. Proteins with a higher proportion of aromatic amino acids will absorb UV light more
effectively per molecule compared to proteins with a lower proportions of these amino acids.
Thus, this method of measuring protein concentration is limited to proteins which their
relationship between absorbance at ODMAX and their concentration is already known.

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Appendix A: Metric units

Table A1. Metric units and prefixes. a) Commonly used units for various measurements. b)
Standardized prefixes assigned to an extremely wide range of numbers. Different prefixes are
commonly used in various aspects of molecular biology. For example, sizes of genomes are
typically in the giga and mega base-pairs. Concentrations and volumes of reagents are commonly
within milli and micro range. Sizes of macromolecules are in the nano and pico range.

a) b)
Decimal Fractions
Symbo Facto
Fraction Arithmetic Symbol Term (prefix) Letter Abbreviation
(prefix) Prefix l r
Thousandth 10-3 milli m yotta Y 1024
Millionth 10-6 micro µ zetta Z 1021
Billionth 10-9 nano n exa E 1018
Trillionth 10-12 pico p
peta P 1015
Molar Concentrations tera T 1012
Mole Fraction Name Letter Abbreviation Amount per Liter giga G 109
1M molar M 1 mole mega M 106
10-3 M millimolar mM 1 millimole kilo k 103
10-6 M micromolar µM 1 micromole hecto h 102
10-9 M nanomolar nM 1 nanomole
deka da 101
Mass deci d 10-1
Name Abbreviation Fraction of a gram centi c 10-2
kilogram kg 103 milli m 10-3
gram g 1 micro u 10-6
milligram mg 10-3
nano n 10-9
microgram µg 10-6
nanogram ng 10-9 pico p 10-12
picogram pg 10-12 femt
o f 10-15
Volume atto a 10-18
Name Abbreviation Fraction of a Liter zepto z 10-21
Liter L 1
milliliter mL 10-3
yocto y 10-24
microliter µL 10-6

Length
Name Abbreviation Fraction of a meter
Kilometer km 103
Meter m 1
Centimeter cm 10-2
Millimeter mm 10-3
Micrometer µm 10-6
Nanometer nm 10-9
Angstrom (not metric) Å 10-10

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Appendix B: Vortex
A vortex is used to mix solutions vigorously (Figure A1). It's operation is simple:
1. Flip the selector to "ON" for continuous motion. Flip the selector to "Touch" to turn on the
vortex only when samples are pressed onto the shaking platform.
2. Speed dial to set speed of vortex.
3. Shaking platform. Press samples onto this platform to activate vortex when it is set to
"Touch" mode.

Figure A1. A benchtop vortex. Numbers correspond to description in the text above.

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Appendix C: Microcentrifuge
A benchtop microcentrifuge, which the '1.5 mL microcentrifuge tubes' are named after, are used
to centrifuge 1.5 mL tubes. This can be used to pellet large particles inside the tube and are also
useful to collect solutions at the bottom of tube after mixing. A benchtop microcentrifuge does
not produce as much centrifugal force as larger floor centrifuges and are relatively safe to operate
(mishandling larger centrifuges could lead to lethal accidents). However, the smaller centrifuges
still should be handled with care (Figure A2).
1. Press the 'Open' button to open the lid. Remove the cover of the rotor and place samples
in a balanced configuration. You MUST place the rotor cover back in place. Close the lid.
2. Use time dial to set the duration of centrifugation.
3. Use speed dial to set the speed of centrifugation.
4. Use the rpm/rcf dial to select the unit of centrifugal force (or, the 'speed').
• RPM = Rotations Per Minute. This sets the centrifugal force by choosing how many
times the rotor rotates per minute.
• RCF = Relative Centrifugal Field. This sets the centrifugal force by choosing the
amount of 'extra weight' used to pull samples towards the circumference of the
rotor, relative to the gravity of the earth (1 g). For example, rcf of 1000x g means
that samples will experience a pulling force 1000 times the force of gravity.
5. Press the start/stop button to start the centrifuge. Press it again to stop it prematurely in
case of emergency (such as hearing unexpected noises, or seeing the centrifuge shake and
move).
• Alternatively, press and hold the 'short' button to make the rotor spin at max speed.
The rotor will spin as long as you are holding the button. This function is useful for
brief spins to 'pull down solutions to the bottom'.

Figure A2. Benchtop microcentrifuge. a) This microcentrifuge is set to spin samples at 14000 rpm
for 3 minutes. b) The microcentrifuge rotor with its cover removed. This rotor has 18 spots to put
in 1.5 mL microcentrifuge tubes. In this picture, two tubes are placed in a balanced configuration
(assuming that these tubes contain equal amount of solution).

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Appendix D: Spectrophotometry
Many compounds absorb ultraviolet or visible light. Colorimetry measures the amount of light
absorbed as it passes through the colored solution, and turbidimetry measures light absorption by
particles in suspended in a solution.

Colorimetry and Turbidimetry can be used to determine the concentration of colored chemicals
and particles in suspension, respectively. The light absorbed by a colored solution (the optical
density, or OD) or by particulate suspension is proportional to the concentration of the light
absorbing material (Beers-Lambert Law).

Photoelectric colorimeters and spectrophotometers are invaluable tools in the laboratory. A highly
simplified diagrammatic representation of the photoelectric colorimeter is shown in Figure A3.
The light from the lamp passes through a filter. The filter is determined by the properties of the
solution being measured. The filtered monochromatic light passes through a cuvette containing
the “blank” solution (100% transmission = 0% absorbance) or the sample to be measured. The
light transmitted through strikes a photo cell that generates a current proportional to the intensity
of the transmitted light. In the diagram, a beam of monochromatic light, of radiant power P0,
directed at the sample solution is transmitted through the solution and now has radiant power P.
Thus, light absorption has taken place in the solution.

Millimeter or Galvanometer, combined

with resistors and transformers to give
P0 P measurement
Slit

Slit
Monochromatic

Photocell
filter
Light source

Sample or Blank

Figure A3. A simplified, schematic diagram of a photoelectric colorimeter.

The amount of radiation absorbed may be measured in a number of ways:

Transmittance, T = P / P0
% Transmittance, % T = 100 (T)
Absorbance,
OD = log10 P0 / P
OD = log10 1 / T
OD = log10 100 / % T
OD = 2 - log10 % T [also stated as: %T = antilog (2.00 – OD)]

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This equation, OD = 2 - log10 % T, is worth noting because it allows you to easily calculate
absorbance (optical density) from percentage transmittance data.

Percent transmittance plotted against concentration produces a curved line. However, plotting
optical density against concentration gives a straight line (theoretically). This allows inferences
about the concentration of the unknown solution by comparing with a single standard solution.

Concentration of Unknown = OD of unknown * concentration of standard / OD of standard

At high concentrations, solutions depart from this linear relationship. Therefore at high
concentrations (OD > 1) should be diluted.

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Appendix E: BIO-RAD SmartSpec Plus Spectrophotometer
A benchtop spectrophotometer is a standard instrument in molecular biology labs due to its
usefulness and ease of operation (Figure A4). The intensities of light-absorbing molecules can be
measured directly which gives an insight to the concentration of those samples.

Note that the 'colours' absorbed by of these molecules are not always visible light. DNA and
proteins absorb light in the ultraviolet range. Also, 'colour-absorbing molecules' do not need to be
macromolecules. For example, number of bacterial cells in a liquid culture can be determined by
measuring absorbance of the culture at around 600 nm (discussed further in Lab 3).

Figure A4. BIO-RAD SmartSpec Plus Spectrophotometer. A quick-start guide is available, pulled
out from the bottom of the instrument.

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