Molecular Biology
Technical Skills
Skills
 Micropipetting
 Preparing
solutions
 Working with concentrations
 Dilutions
 Amounts
 Agarose gel electrophoresis
Micropipetting- Measuring
small volumes
 Allows
1

to measure microliters (µL)
000 X less than 1 milliliter
2-20 µL
Max. 0.02 mL
50-200 µL
0.2mL
100-1000 µL
1mL
Setting the volume- P20
Tens (0, 1=10 or 2=20)
Units (0-9)
Decimal (1-9 = 0.1-0.9)
Setting the volume- P200
Hundreds (0, 1=100 or 2=200)
Tens (0, 1-9=10-90)
Units (1-9)
Setting the volume- P1000
Thousands (0, 1=1000)
Hundreds (0, 1-9=100-900)
Tens (0, 1-9=10 - 90)
Using the micropipettor
Step 1
Insert tip
Step 2
Press plunger
up to first stop
Step 3
Insert tip in solution to be
drawn
Step 4
Draw up sample by
slowly releasing plunger
Step 5
Withdraw pipettor
Dispensing
Start dispensing
1st stop =Dispense 2nd stop = Expel
Guidelines for optimal
reproducibility
 Use
pipettor whose volume is closest to the
one desired
 Consistent SPEED and SMOOTHNESS to
press and release the PLUNGER
 Consistent IMMERSION DEPTH
 3-4mm
 AVOID
below surface
air bubbles
 NEVER go beyond the limits of the pipettor
Preparing Solutions
Definitions
 Solution
 Mixture
of 2 or more substances in a single
phase
 Solutions are composed of two
constituents
 Solute
Part that is being dissolved or diluted – Usually
smaller amount
 Solvent
(OR Diluent)
Part of solution in which solute is dissolved – Usually
greater volume
Concentrations

Concentration = Quantity of solute
Quantity of solution (Not solvent)

Three basic ways to express concentrations:



Molar concentration (Molarity)
Percentages
Mass per volume
Molarity
#
of Moles of solute/Liter of solution
 Mass
of solute/MW of solute = Moles of solute
 Moles
of solute/vol. in L of solution = Molarity
Percentages
 Percentage
concentrations can be
expressed as either:
 V/V
– volume of solute/100 mL of solution
 W/W – weight of solute/100g of solution
 W/V – Weight of solute/100mL of solution
 All
represent fractions of 100
Percentages
(Cont’d)
 %V/V
 Ex.
4.1L solute/55L solution =7.5%
 Must
have same units top and bottom!
 %W/V
 Ex.
16g solute/50mL solution =32%
 Must
have units of same order of magnitude
top and bottom!
%
W/W
 Ex.
1.7g solute/35g solution =4.9%
 Must
have same units top and bottom!
Dilutions
Reducing a Concentration
A Fraction
Dilutions
 Dilution
= making weaker solutions
from stronger ones
 Example: Making orange juice from
frozen concentrate. You mix one can of
frozen orange juice with three (3) cans
of water.
Dilutions (cont’d)
 Dilutions
are expressed as the volume
of the solution being diluted per the
total final volume of the dilution
 In the orange juice example, the
dilution would be expressed as 1/4, for
one can of O.J. to a TOTAL of four cans
of diluted O.J. When saying the
dilution, you would say, in the O.J.
example: “one in four”.
Dilutions (cont’d)
 Another
example:
 If you dilute 1 ml of serum with 9 ml of
saline, the dilution would be written
1/10 or said “one in ten”, because you
express the volume of the solution
being diluted (1 ml of serum) per the
TOTAL final volume of the dilution (10
ml total).
Dilutions (cont’d)
 Another
example:
 One (1) part of concentrated acid is
diluted with 100 parts of water. The
total solution volume is 101 parts (1
part acid + 100 parts water). The
dilution is written as 1/101 or said “one
in one hundred and one”.
Dilutions (cont’d)
 Notice
that dilutions do NOT have units
(cans, ml, or parts) but are expressed
as one number to another number
 Example:
1/10 or “one in ten”
Dilutions (cont’d)
 Dilutions
are always expressed with the
original substance diluted as one (1). If
more than one part of original
substance is initially used, it is
necessary to convert the original
substance part to one (1) when the
dilution is expressed.
Dilutions (cont’d)
Example:
Two (2) parts of dye are diluted with eight (8) parts
of diluent (the term used for the diluting solution).
The total solution volume is 10 parts (2 parts dye + 8
parts diluent). The dilution is initially expressed as
2/10, but the original substance must be expressed
as one (1). To get the original volume to one (1),
use a ratio and proportion equation, remembering
that dilutions are stated in terms of 1 to something:
______2 parts dye
=
___1.0___
10 parts total volume
x
2x
=
10
x
=
5
The dilution is expressed as 1/5.
Dilutions (cont’d)
The dilution does not always end up in whole numbers.
Example:
Two parts (2) parts of whole blood are diluted with
five (5) parts of saline. The total solution volume is
seven (7) parts (2 parts of whole blood + 5 parts
saline). The dilution would be 2/7, or, more
correctly, 1/3.5. Again, this is calculated by using the
ratio and proportion equation, remembering that
dilutions are stated in terms of 1 to something:
__2 parts blood_____
=
___1.0___
7 parts total volume
x
2x
=
7
x
=
3.5
The dilution is expressed as 1/3.5
What Does This Mean??
 If
a solution has a 1/10 dilution the
fraction represents 1 part of the sample
being diluted added to 9 parts of diluent
for a total of 10 parts.
 If this solution was prepared to a final
volume of 110 mL, what volumes of
solute and what volume of solvent have
to be used?
 In other words, what is the volume of 1
part and of 9 parts?
Dilution Factor

EXAMPLE: What is the dilution factor if you
add 0.1 mL aliquot of a specimen to 9.9 mL
of diluent?


The final volume is equal to the aliquot volume
PLUS the diluent volume:
0.1 mL + 9.9 mL = 10 mL
The dilution factor is equal to the final volume
divided by the aliquot volume:
10 mL/0.1 mL = 100X dilution factor
Practice Problem
 What
is the dilution factor when 0.2 mL
is added to 3.8 mL of diluent?
Serial Dilutions
 If
a 1/8 dilution of the stock solution is
made followed by a 1/6 dilution what is
the final dilution?
 The final dilution is: 1/8 x 1/6 = 1/48
Dilutions
 Means
to reduce a concentration
 Dilution:
A fraction of the dilution factor
Dilution factor = Conc. I have
Conc. I want
Ex. You have a solution at 25 mg/ml and
wish to obtain a solution of 5mg/ml
Dilution factor = 25mg/mL = 5X
5mg/mL
Dilution = 1/the dilution factor = 1/5 = 1 part/5 parts Total
Example
 How
would you prepare 25mL of a 2mM
solution from a 0.1M stock?
Quantities
 Quantities
are equal to amounts NOT
concentrations!
 Ex
1.
 Two
apples per bag = a concentration
 Two apples = an amount
 Ex
2.
 10g
per 100 mL = a concentration
 10g = an amount
From concentrations to amounts
 The
concentration indicates the amount
in a given volume
 Ex.
1mM = 1millimole per each liter
 Therefore the amount in 1 L is 1 millimole
 What volume of solution would you need
to have 0.05 millimoles?
Agarose Gel Electrophoresis
 Separates
single stranded or double
stranded nucleic acid molecules
according to their size and their
conformation
 Separates
fragments between 100pb and
10 Kbp
 Resolving power between fragments
≥100pb
Migration on an Agarose Gel
Top (-)
Well
Linear
Relaxed
Direction of
migration
Supercoiled
Bottom (+)
What can be determined from an
electrophoresis on an agarose gel?
 Is
there DNA
 How many conformations
 How many fragments
 The
size of the fragments
 Total size of nucleic acid molecule
 The number of cuts
 Linear?
 Circular?
Migration Profile on Agarose
Resolution
Log of the size
Resolution
1.0%
1.5%
Migration distance
Determining sizes
100,000
Fingerprinting Standard Curve: Semi-log
Distance (mm)
23,000
11.0
9,400
13.0
6,500
15.0
4,400
18.0
2,300
23.0
2,000
24.0
10,000
Size, base pairs
Size (bp)
B
1,000
100
0
5
10
15
Distance, mm
20
A
25
30