The Basic Skills of the
Biotechnology Workplace
Chapter 3
Learning Outcomes
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Determine the most appropriate tool for measuring specific volumes of
masses
Describe how to select, set, and use a variety of micropipets within their
designated ranges to accurately measure small volumes
Convert between units of measure using the B <- -> S rule and appropriate
conversion factors
Recognize the different expressions for units of concentration measurements
and use their corresponding equations to calculate the amount of solute
needed to make a specified solution
Describe what pH is and why it is important in solution preparation
3.1 Measuring Volumes in a Biotechnology Facility
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Volume is a measurement of the amount of space
something occupies
Volume is measured in
• Liters (L)
• Milliliters (mL)
• Microliters (mL)
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Different tools are used to measure volume
• Graduated cylinder
• Pipet
• Micropipet
Converting Units
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Often volumes are measured in one unit of measurement and
reported in another
Converting between metric units
Conversion factor
To measure volumes larger than 10 milliliters, technicians usually use
a graduated cylinder
Pipets are available that measure volumes
between 0.1 mL and 50 mL. Shown from left
to right are 25-, 10-, 5-, and 1-mL pipets.
Reading a graduated cylinder. Before using
a graduated cylinder, make sure you know the
total volume it will hold and the value of each
of the graduations. In the lab, common
graduated cylinders include 10 mL, 25 mL, 100
mL, 250 mL, 500 mL, and 1 L.
Using Pipets
• Measuring units smaller than 10
mL requires a pipet.
• “Never mouth pipet!”
Using Micropipets
• When measuring tiny
volumes, less that 1 mL, a
micropipet is used.
• A micropipet has four parts
• Plunger button
• Ejector button
• Volume display
• Dispensing tip
Picking and Using the
Appropriate Micropipet
• P-100 or P-200 micropipet
• P-10 or P-20 micropipet
• P-1000 micropipet
Labeled micropipet. Learning
to use each part of a micropipet
correctly is essential. On the
micropipet shown, the plunger
has two “stops.” Pressing to the
first stop evacuates air to the
volume in the display. Pressing to
the second stop evacuates that
volume plus another 50% or so.
To ensure accurate measurement,
feel the difference between the
first and second stop before using
the pipet. Inaccurate
measurement could waste costly
reagents and cause invalid
experiment results.
P-100 Micropipet. This
micropipet will measure volumes
as small as 10 mL and has
precision to 0.2 mL.
P-10 Micropipet. P10 micropipets are
common in
biotechnology labs. A
P-10 micropipet will
measure volumes as
small as 0.5 mL and
has precision to 0.02
mL. A P-10 uses tiny
tips that are usually
white.
P-1000 Micropipet.
A P-1000 micropipet
will measure up to
1000 mL, or 1 mL,
and uses large tips
that are usually blue
or white in color.
A multichannel pipet allows several samples to be measured at the same time, a feature
that saves time during an experiment with multiple replications and repetitive pipeting.
Vocabulary
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Volume – a measurement of the amount of space something occupies
Mass – the amount of matter (atoms and molecules) an object contains
Liter – abbreviated “L”; a unit of measure for volume, approximately equal to a quart
Milliliter – abbreviated “mL”; a unit of measure for volume; one one-thousandth of a liter (0.001
L) or about equal to one-half teaspoon
Microliter – abbreviate “mL”; a unit of measure for volume; equivalent to one-thousandth of a
milliliter or about the size of the tiniest teardrop
Graduated cylinder – a plastic tube with marks (or graduations) equally spaced to show volumes;
measurements are made at the bottom of the meniscus, the lowest part of the concave surface of
the liquid in the cylinder
Pipet – an instrument usually used to measure volumes between 0.1 mL and 50 mL
Micropipet – an instrument used to measure very tiny volumes, usually less than a milliliter
Unit of measurement – the form in which something is measured (g, mg, mg, L, mL, mL, km, cm,
etc.)
Conversion factor – a number (a fraction) where the numerator and denominator are equal to
the same amount; commonly used to convert from one unit to another
Metrics conversion table – a chart that shows how one unit of measure relates to another (for
example, how many milliliters are in a liter)
3.1 Review Questions
1.
What instrument would you use to measure and dispense the
following volumes? Pick the instrument that is likely to give you the
least error for each measurement.
23.5 mL
2.
6.5 mL
125 mL
7mL
2.87 mL 555mL
Convert the following units to the requested unit:
1.7 L = _____ mL
235.1 mL = _____ mL
2.37 mL = _____ mL
3.
What numbers should be dialed into a P-10 display if a volume of
3.7 mL is to be measured?
4.
What instrument should be used if a technician wants to fill 40 sets
of 16 tubes all with identical volumes?
3.2 Making Solutions
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Solution preparation is one of the most essential
skills of a biotechnology lab employee.
Solutions are mixtures in which one or more
substances are dissolved in another substance.
Solid solutes are measured on balances or scales.
Concentration is measured in several ways:
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Mass/volume
Volume/volume
% mass/volume
Molarity
Normality
Most analytical
balances measure
down to milligrams,
even though they
usually report in
grams.
Vocabulary
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Positive displacement micropipet – an instrument that is generally used to pipet small
volumes of viscous (thick) fluids
Solution – a mixture of two or more substances where one (solute) completely dissolves in
the other (solvent)
Aqueous – describing a solution in which the solvent is water
Solute – the substance in a solution that is being dissolved
Balance – an instrument that measures mass
Weight – the force exerted on something by gravity; at sea level, it is considered equal to
the mass of an object
Gram – abbreviated “g”; the standard unit of mass, approximately equal to the mass of a
small paper clip
Solvent – the substance that dissolves the solute
Molarity – a measure of concentration that represents the number of moles of a solute in a
liter of solution (or some fraction of that unit)
Normality – a measurement of concentration generally used for acids and bases that is
expressed in gram equivalent weights of solute per liter of solution; represents the amount
of ionization of an acid or base
3.2 Review Questions
1.
What instrument should be used to measure and dispense the
following solutes? Choose the instrument that is likely to give you
the least error for each measurement.
3.5 g of salt
6.5 mg of DNA
12.5 g of gelatin
2.
What happens to the ratio of solute molecules to solvent as a
solution becomes more concentrated?
3.
Which of the following are concentration units?
mi/hr
4.
g/mL
mM
°F/°C
Describe how glassware should be prepared before using it to
prepare or store solutions.
3.3 Solutions of Given Mass/Volume Concentrations
Mass/Volume Solution. Solvent is added until a volume of 10 mL is reached. A
protein solution that has a concentration of 1 g/mL is considered fairly concentrated.
Making Mass/Volume Solutions
Mass/Volume Concentration Equation
____ g/mL X ____ mL
concentration
volume
desired
desired
=
____ g of solution
to be weighed out,
dissolved in the solvent
3.3 Review Questions
1.
Which of the following are mass/volume concentration units?
mg/mL
g/mg
L/mg
mg/mL
g/l
2.
What mass of the protein, gelatin, is needed to make 0.5 L of a 3
g/L gelatin solution?
3.
What mass of sugar is need to make 25 mL of a 25 mg/mL sugar
solution?
4.
What mass of salt is needed to make 150 mL of a 100 mg/mL salt
solution? Describe how the solution is prepared.
3.4 Solutions of Differing % Mass/Volume Concentrations
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A percentage represents something that is part of 100.
Mass/Volume Concentration Equation
____ %
percent value
____
decimal
value
X
=
____
decimal value of the g/mL
____
=
total volume
desired (mL)
____ g of solute to
be measured and
added to the volume
desired of solvent
Vocabulary
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Percentage – a proportion of something out of 100 parts,
expressed as a whole number
3.4 Review Questions
1.
What is the decimal equivalent of the following percentages?
10%
15%
25%
2%
1.5%
0.5%
2.
What mass of gelatin (a protein) is needed to make 0.5 L of a 3%
gelatin solution?
3.
What mass of sugar is needed to make 25 mL of a 2.5% sugar
solution?
4.
What mass of salt is needed to make 150 mL of a 10% salt
solution? Describe how the solution is prepared.
3.5 Solutions of Differing Molar Concentrations
Molarity Concentration Equation
volume
wanted (L) X
molarity
desired
(mol/L)
molecular
X weight of the
solute (g/mol)
=
the number of grams to be
dissolved in solvent, up to
the total volume of
solution desired
Periodic Table. The Period Table of Elements shows the elements (atoms) found
in compounds (molecules). Each element is listed along with the atomic weight
(mass) of each atom in the element. A NaCl molecule has a molecular weight of
about 58.5 amu (atomic mass units) because the Na atom weighs about 23 amu,
and the Cl atom weighs about 35.5 amu. Together, in the NaCl molecule, the
atoms total approximately 58.5 amu. The mass of a hydrogen atom equals 1 amu.
This instrument is a mass spectrometer. Scientists use it to determine the
molecular weight of a compound. A “mass spec” can also determine if a
sample is contaminated with molecules of different molecular weights.
Vocabulary
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Mole – the mass, in grams, of 6 X 1023 atoms of molecules of a given substance;
one mole is equivalent to the molecular weight of a given substance, reported in
grams
Molecular weight – the sum of all the atomic weights of the atoms in a given
molecule
amu – abbreviation of atomic mass unit; the mass of a single hydrogen atom
Mass spectrometer – an instrument that is used to determine the molecular
weight of a compound
3.5 Review Questions
1.
What is the molecular weight of each of the following compounds?
NaOH
MgCl2
MgO
HCl
2.
What mass of NaCl is needed for 0.5 L of a 0.5 M NaCl solution?
3.
What mass of MgO is needed for 200 mL of a .025 M MgO solution?
4.
What mass of sodium hydroxide (NaOH) is needed to make 750 mL
of a 125 mM NaOH solution? Describe how to prepare the solution.
3.6 Dilutes of Concentrated Solutions
Concentrating 1 L Solution.
Many chemical and biological
reagents are purchased in
concentrated form. Concentrated
solutions can be prepared initially
with a greater amount of solute to
solvent, or a solution can be
concentrated by removing water. A
diluted solution can be prepared by
adding solvent to a concentrated
one.
Diluting a 100 mg/mL Stock Solution to 1 mg/mL.
Vocabulary
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Dilution – the process in which solvent is added to make a solution less concentrated
Stock solution – a concentrated form of a reagent that is often diluted to form a
“working solution”
Buffer – a solution that acts to resist a change in pH when the hydrogen ion
concentration is changed
TRIS – a complex organic molecule used to maintain the pH of a solution
TAE buffer – a buffer that is often used for running DNA samples on agarose gels in
horizontal gel boxes; contains TRIS, EDTA, and acetic acid
3.6 Review Questions
1.
How do you prepare 40 mL of a 2 mg/mL protein solution from 10
mg/mL protein solution?
2.
How do you prepare 200 mL of 2X enzyme buffer from 10X enzyme
buffer solution?
3.
How do you prepare 500 mL of 10 mM NaCl solution from 5 mM NaCl
solution?
4.
How do you prepare 3 L of 1X TAE buffer from 50X TAE buffer stock
solution?
Questions and Comments?