The Basic Skills of the Biotechnology Workplace

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The Basic Skills of the Biotechnology Workplace
Chapter 3
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Learning Outcomes

• 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 lt-
  -gt 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
• Define the term buffer and calculate a specified
  dilution from a concentrated stock solution or
  buffer

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3.1 Measuring Volumes in a Biotechnology Facility

• Volume is a measurement of the amount of space
  something occupies
• Volume is measured in
• Liters (L)
• Milliliters (mL)
• Microliters (mL)
• Different tools are used to measure volume
• Graduated cylinder
• Pipet
• Micropipet

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Converting Units

• 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

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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.
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.
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Using Pipets
Picking and Using the Appropriate Micropipet

• Measuring units smaller than 10 mL requires a
  pipet.
• Never mouth pipet!

• P-100 or P-200 micropipet
• P-10 or P-20 micropipet
• P-1000 micropipet

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

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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.
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P-10 Micropipet. P-10 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.
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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.
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Vocabulary

• Volume a measurement of the amount of space
  something occupies
• Mass 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)

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3.1 Review Questions

• 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 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
• What numbers should be dialed into a P-10 display
  if a volume of 3.7 mL is to be measured?
• What instrument should be used if a technician
  wants to fill 40 sets of 16 tubes all with
  identical volumes?

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3.2 Making Solutions

• 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
• Mass/volume
• Volume/volume
• mass/volume
• Molarity
• Normality

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Most analytical balances measure down to
milligrams, even though they usually report in
grams.
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Vocabulary

• 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

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3.2 Review Questions

• 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
• What is the relation of solute to solvent as a
  solution becomes more concentrated?
• Which of the following are concentration units?
• mi/hr g/mL mM F/C
• 4. Describe how glassware should be prepared
  before using it to prepare or store solutions.

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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 mg/mL is considered
fairly concentrated.
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Making Mass/Volume Solutions
Concentration in Mass/Volume Units
____ g/mL X ____ mL ____ g of
solution concentration volume to be weighed
out, desired desired dissolved in the solvent
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3.3 Review Questions

• Which of the following are mass/volume
  concentration units?
• mg/mL g/mg L/mg mg/mL g/l
• What mass of the protein, gelatin, is needed to
  make 0.5 L of a 3 g/L gelatin solution?
• What mass of sugar is need to make 25 mL of a 25
  mg/mL sugar solution?
• What mass of salt is needed to make 150 mL of a
  100 mg/mL salt solution? Describe how the
  solution is prepared.

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3.4 Solutions of Differing Mass/Volume
Concentrations

• A percentage represents something that is part of
  100.

Concentration Mass/Volume Equation
____ ____ percent
value decimal value of the g/mL
____ X ____
____ g of solute to decimal total volume be
measured and value desired (mL) added to the
volume desired of solvent
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Vocabulary

• Percentage a proportion of something out of 100
  parts, expressed as a whole number

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3.4 Review Questions

• What is the decimal equivalent of the following
  percentages?
• 10 15 25 2 1.5 0.5
• What mass of gelatin (a protein) is needed to
  make 0.5 L of a 3 gelatin solution?
• What mass of sugar is needed to make 25 mL of a
  2.5 sugar solution?
• What mass of salt is needed to make 150 mL of a
  10 salt solution? Describe how the solution is
  prepared.

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3.5 Solutions of Differing Molar Concentrations
Molarity Concentration Equation
volume molarity molecular the number of grams
to be wanted (L) X desired X weight of the
dissolved in solvent, up to (mol/L) solute
(g/mol) the total volume of solution
desired
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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 58.4 atu (atomic mass units) because the
Na atom weighs about 23 amu, and the Cl atom
weighs about 35.4 amu. Together, in the NaCl
molecule, the atoms total approximately 58.4 amu.
The mass of a hydrogen atom equals 1 amu.
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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.
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Vocabulary

• 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

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3.5 Review Questions

• What is the molecular weight of each of the
  following compounds?
• NaOH MgCl2 MgO HCl
• What mass of NaCl is needed for 0.5 L of a 0.5 M
  NaCl solution?
• What mass of MgO is needed for 200 mL of a .025 M
  MgO solution?
• What mass of sodium hydroxide (NaOH) is needed to
  make 750 mL of a 125 mM NaOH solution? Describe
  how to prepare the solution.

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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 the solution.
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Diluting a 100 mg/mL Stock Solution to 1 mg/mL.
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Vocabulary

• 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

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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?

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Questions and Comments?