Biotech Chapter Three Lecture- Basic Lab Skills

The Basic Skills of the
Biotechnology Workplace
Chapter 3

Learning Outcomes

 Determine the most appropriate tool for
measuring specific volumes of masses

Learning Outcomes

 Describe how to select, set, and use a
variety of micropipets within their designated
ranges to accurately measure small volumes

Learning Outcomes

 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

Learning Outcomes
 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

Learning Outcomes
 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


Volume is a measurement of the amount of space
something occupies


something occupies

Volume is measured in
• Liters (L)
• Milliliters (mL)
• Microliters (µL)


something occupies

Volume is measured in
• Liters (L)
• Milliliters (mL)
• Microliters (µL)
Different tools are used to measure volume
• Graduated cylinder
• Pipet
• Micropipet

Converting Units
Often volumes are measured in one unit of
measurement and reported in another

Converting Units
•Converting between metric units

Converting Units
move decimal point

Converting Units
move decimal point
B S

Converting Units
move decimal point
B S

•Use a conversion factor for non-metric

To measure volumes larger
than 10 milliliters, technicians
usually use a graduated
cylinder
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.

A volumetric ﬂask is a
more precise choice

will only measure one
quantity

Using Pipets
•Measuring units smaller
than 10 mL requires a
pipet.
• “Never mouth pipet!”

25-, 10-, 5-, and 1-
mL SEROLOGICAL VOLUMETRIC
pipets pipets
= better accuracy
= pretty accurate

Using Micropipets
When measuring tiny volumes, less that 1 mL,
a micropipet is used.

Picking and Using the Appropriate Micropipet

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

A micropipet has four parts
• Plunger button
• Ejector button
• Volume display
• Dispensing tip

Learning to use each part of a
micropipet correctly is essential.

Inaccurate measurement could
waste costly reagents and cause
invalid experiment results.

On the micropipet shown, the plunger has
two “stops.”

two “stops.”

Pressing to the ﬁrst stop evacuates air to
the volume in the display. Used for
drawing liquid.

two “stops.”

drawing liquid.
Pressing to the second stop evacuates
that volume plus another 50% or so. Used
to dispense.

two “stops.”

drawing liquid.
Pressing to the second stop evacuates
that volume plus another 50% or so. Used
to dispense.
To ensure accurate measurement, feel the
difference between the ﬁrst and second
stop before using the pipet.

P-1000 Micropipet.
100- 1000 µL (1 mL)
large tips are usually blue
or white in color.

P-100 Micropipet.
10 - 100 µL
precision to 0.2 µL
yellow tips

P-10 Micropipet.
P-10 micropipets are
common in biotechnology
labs.
0.5 - 10.0 µL
precision to 0.02 µL.
tiny tips are usually white

Picking and Using the Appropriate Micropipet

Always use the
smallest device
possible.

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

• Volume – a measurement of the amount of space something
occupies

Vocabulary

occupies
• Mass – the amount of matter (atoms and molecules) an object
contains

Vocabulary

occupies
contains
• Liter – abbreviated “L”; a unit of measure for volume, approximately
equal to a quart

Vocabulary

occupies
contains
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

Vocabulary

occupies
contains
equal to a quart
teaspoon
• Microliter – abbreviate “µL”; a unit of measure for volume;
equivalent to one-thousandth of a milliliter or about the size of the
tiniest teardrop

Vocabulary

occupies
contains
equal to a quart
teaspoon
• Microliter – abbreviate “µL”; 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

Vocabulary

• Pipet – an instrument usually used to measure volumes
between 0.1 mL and 50 mL

Vocabulary

• Micropipet – an instrument used to measure very tiny
volumes, usually less than a milliliter

Vocabulary

• Unit of measurement – the form in which something is
measured (g, mg, µg, L, mL, µL, km, cm, etc.)

Vocabulary

• 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

Vocabulary

• 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

0 0 0

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.

0 0 0

23.5µL 6.5mL 125mL 7µL 2.87mL 555µL

0 0 0

23.5µL 6.5mL 125mL 7µL 2.87mL 555µL
2. Convert the following units to the requested unit:

0 0 0

23.5µL 6.5mL 125mL 7µL 2.87mL 555µL

1.7 L = _____ mL
235.1 µL = _____ mL

0 0 0

23.5µL 6.5mL 125mL 7µL 2.87mL 555µL

1.7 L = _____ mL
235.1 µL = _____ mL
2.37 mL = _____ µL

0 0 0

23.5µL 6.5mL 125mL 7µL 2.87mL 555µL

1.7 L = _____ mL
235.1 µL = _____ mL
2.37 mL = _____ µL
3. What numbers should be dialed into a P-10 display if a
volume of 3.7 µL is to be measured? 0 0 0

23.5µL 6.5mL 125mL 7µL 2.87mL 555µL

1.7 L = _____ mL
235.1 µL = _____ mL
2.37 mL = _____ µL
3. What numbers should be dialed into a P-10 display if a
volume of 3.7 µL is to be measured? 0 0 0
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
• 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.

• Solid solutes are measured on balances or
scales.
• Concentration is measured in several ways:
•Mass/volume ●Volume/volume
•% mass or % volume ●Molarity
•Normality

To make any solution-
1) calculate quantity of solute needed for desired
amount of solution.
amount of solution x concentration = amount of solute
(if units agree)

amount of solution.
(if units agree)
2) Measure amount of solute

amount of solution.
(if units agree)
3) Add solvent to desired volume

amount of solution.
(if units agree)
4) All measuring devices must be clean (rinsed with
deionized water) and completely dry
OR rinsed with the material to be measured.

amount of solution.
(if units agree)
4) All measuring devices must be clean (rinsed with
deionized water) and completely dry
OR rinsed with the material to be measured.
Dirty glassware will contaminate/increase solute.
Wet glassware will dilute the solution.

3.3 Mass/Volume Solutions
Concentration is measured in several ways:
Mass/volume

Mass/volume
•Do concentration calculations to determine
the amount of solute required to produce a
certain volume.
Ex. 200ml of a 5g/L solution
requires 5g/L x .2L = 1g solute

Mass/volume
certain volume.

•Measure desired mass of solute
using a balance

Mass/volume
certain volume.

•Measure desired mass of solute
using a balance
•Add solvent to desired volume

Mass/Volume Solution. Solvent is added to 10g protein
until a volume of 10 mL is reached. A protein solution that has a
concentration of 1 g/mL is considered fairly concentrated.

An electronic
balance is good for
measuring 1-200
grams.

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

1. What instrument should be used to measure and
dispense the following solutes?

3.5 g of salt
6.5 mg of DNA
12.500 g of gelatin


3.5 g of salt
6.5 mg of DNA
12.500 g of gelatin

2. What happens to the ratio of solute molecules to
solvent as a solution becomes more concentrated?


3.5 g of salt
6.5 mg of DNA
12.500 g of gelatin


3. Which of the following are concentration units?

mi/hr
g/mL
mM
°F/°C


3.5 g of salt
6.5 mg of DNA
12.500 g of gelatin


3. 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.

1. Which of the following are mass/volume
concentration units?

g/mL
g/mg
L/mg
µg/µL
g/l
m


g/mL
g/mg
L/mg
µg/µL
g/l
m
2. What mass of the protein, gelatin, is needed to
make 0.5 L of a 3 g/L gelatin solution?


g/mL
g/mg
L/mg
µg/µL
g/l
m
3. What mass of sugar is need to make 25 mL of
a 25 mg/mL sugar solution?


g/mL
g/mg
L/mg
µg/µL
g/l
m
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 µg/mL salt solution? Describe how
the solution is prepared.

3.4- v/v and w/w solutions
Volume/volume

Volume/volume
1) calculate solute amount
2 liters of a 20ml/L solution requires
40ml of solute (2L x 20ml/L = 40ml)

Volume/volume

2) Measure solute with cylinder or pipet.

Volume/volume

2) Measure solute with cylinder or pipet.

3) Add solvent to ﬁnal volume (1960ml)

% mass or % volume

% mass or % volume

A %(v/v) solution is made the same as a volume/
volume solution, but concentration is expressed as
a percent.

% mass or % volume

a percent.

A 20ml/L solution is 20ml/1000ml, which is 0.02
or 2%(v/v).

% mass or % volume

a percent.

A 20ml/L solution is 20ml/1000ml, which is 0.02
or 2%(v/v).

A %(w/w) solution is very similar but solute &
solvent must be weighed.


1. What is the decimal equivalent of the following
percentages?

10%
15%
25%
2%
1.5%
0.5%


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?


percentages?

10%
15%
25%
2%
1.5%
0.5%


3. What mass of sugar is needed to make 25 mL of a
2.5% sugar solution?


percentages?

10%
15%
25%
2%
1.5%
0.5%


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 Molarity
Molarity = moles/Liter

3.5 Molarity
1) Determine moles of solute required.
Molarity x volume of solution = moles solute
250ml of 2M NaCl needs
0.25L x 2M = 0.5moles NaCl

3.5 Molarity
2) Convert moles to grams (multiply by molar
mass of solute)
0.5mol NaCl x 58.5g/mol = 29.25g NaCl

3.5 Molarity
mass of solute)
3) Mass required amount of solute.

3.5 Molarity
mass of solute)
3) Mass required amount of solute.
4) Add solvent to ﬁnal volume.


1.What is the molecular weight of each of the
following compounds?

NaOH
MgCl2
Mg(OH)2
HCl



NaOH
MgCl2
Mg(OH)2
HCl

2. What mass of NaCl is needed for 0.5 L of a 0.5 M
NaCl solution?



NaOH
MgCl2
Mg(OH)2
HCl

NaCl solution?
3. What mass of MgCl2 is needed for 200 mL of a
0.025 M MgCl2 solution?



NaOH
MgCl2
Mg(OH)2
HCl

NaCl solution?
3. What mass of MgCl2 is needed for 200 mL of a .
025 M MgCl2 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.

Vocabulary
 Solution – a mixture of two or more substances
where one (solute) completely dissolves in the
other (solvent)

Vocabulary
other (solvent)
 Aqueous – describing a solution in which the
solvent is water

Vocabulary
other (solvent)
solvent is water
 Solute – the substance in a solution that is being
dissolved

Vocabulary
other (solvent)
solvent is water
dissolved
 Balance – an instrument that measures mass

Vocabulary
other (solvent)
solvent is water
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

Vocabulary

 Gram – abbreviated “g”; the standard unit of mass,
approximately equal to the mass of a small paper clip

Vocabulary

 Solvent – the substance that dissolves the solute

Vocabulary

 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)

Vocabulary

 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 represents the
amount of ionization of an acid or base.
2M HCl = 2N HCl 2M H2SO4 = 4N H2SO4

Vocabulary

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

20/100 = 0.2 = 20%
 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

 Mass spectrometer – an instrument that is used to
determine the molecular weight of a compound

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.

3.6 Dilutes of Concentrated
Solutions
Many chemical and
biological reagents are
purchased in concentrated
form.

Solutions
Many chemical and
form.
Concentrated solutions can
be prepared initially with a
greater amount of solute to
solvent, or a solution can be
concentrated by removing
water.

Solutions
Many chemical and
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.

Solutions
Concentrationstock x Volumestock
=
Concentrationdiluted x Volumediluted

CsVs = CdVd

Solve for the volume of stock solution needed

Diluting a 100 mg/mL Stock Solution to make 1L of 1 mg/mL.

100 mg/mL x Vs = 1 mg/mL x 1000mL
need 10 ml of stock

Vocabulary
 Dilution – the process in which solvent is added to
make a solution less concentrated

Vocabulary
 Stock solution – a concentrated form of a reagent
that is often diluted to form a “working solution”

Vocabulary
 Buffer – a solution that acts to resist a change in pH
when the hydrogen ion concentration is changed

Vocabulary
 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

1. How do you prepare 40 mL of a 2 mg/mL
protein solution from 10 mg/mL protein
solution?

solution?
2. How do you prepare 200 µL of 2X enzyme
buffer from 10X enzyme buffer solution?

solution?
3. How do you prepare 500 µL of 10 µM NaCl
solution from 5 µM NaCl solution?

solution?
3. How do you prepare 500 µL of 10 µM NaCl
solution from 5 µM NaCl solution?
4. How do you prepare 3 L of 1X TAE buffer from
50X TAE buffer stock solution?

Biotech Chapter Three Lecture- Basic Lab Skills

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