CELL AND MOLECULAR

BIOLOGY LABORATORY
PRELIMINARY PERIOD
EXPERIMENT 1
Use of Micropipettor and
Spectrophotometer
Micropipettor

biology are milliliter (mL) and

microliter (uL)
Micropipettor is a precision
pump with a disposable tip

How to use the Micropipettor

1. The volume of air space in
the barrel is adjusted by
screwing the plunger further in
or out of the piston
2. The volume is displayed on
the digital readout
3. Depressing the plunger will
displace the specified volume
of air from the piston
4. Releasing the plunger creates
a vaccum, which will draw
an equal volume of fluid
into the tip
5. Withdrawn fluid is expelled by
depressing the plunger
- If there are air bubbles,
depress the plunger and
withdraw needed amount of
volume (again)
Types of Micropipettors
- Red
1. range is 0.5-1.0 uL
2. tip: 10 uL

Micropipettor is used to
transfer volumes that are less
than 1mL (small volumes of
DNA and reagents)
Measures as little as one
microliter (uL), one millionth
(10^-6) of a litter
1mL= 0.001L
1uL=0.000001L
1L=1000mL
1L=1000000uL
Most useful units of liquid
measurement in molecular

- Yellow
3. range is 10-100 uL
4. tip: 100 uL
- Blue
5. range is 1000-1000 uL
6. tip: 1000 uL
USING THE MICROPIPETTOR:
Rotate the volume adjustor to
the desired setting- use
specific micropipettor for the
specific volume
Firmly get the tip on the end
of micropipettor tip = the tip

color should correspond to

the micropipettor color
Hold the tube NEARLY at eye
level and hold the tube firmly
between your thumb and
forefinger
DO NOT:
Pipet with the tube in the test
tube rack
Have another person hold the
tube while pipetting
Grasp the tube body not the
lid so that there is greater
control
and
AVOID
CONTAMINATION OF THE
MOUTH OF THE TUBE
Hold the micropipettor in your
palm and wrap your fingers
around the barrel work the
plunger/piston with the thumb
(HOLD
THE
MICROPIPETTOR
almost
vertical when filling it)
Micropipettors
have
two
stops when only getting
the desired volume from the
tube: PRESS ONCE
SECOND STOP: This will
introduce
an
additional
volume of AIR to blow out
any solution remaining in
the tip
When withdrawing liquid,
NO AIR SPACE at the very
end of the tip: if there is air
space: expel the sample back
to the tube and coalesce the
sample by sharply tapping the
tube on the bend top or
pulsing it in a microfuge
EXPELLING SAMPLE: there
is a CAPILLARY ACTION
when the tip of pipet touches
the inside wall of reaction tube
in which the sample will be
emptied

- Depress up to second stop

for excess fluid
TO
PREVENT
CROSSCONTAMINATION
OF
REAGENTS
7. Always
add
appropriate
amounts of single reagent
sequentially to all reaction
tubes
8. Release each reagent drop
onto a new location inside the
wall, near the BOTTOM of the
reaction tube
9. Use a fresh top for each new
reagent
10. Switch tip if contaminated
Spectrophotometer

Light comes from a light

source (tungsten lamp or
delirium)
then
to
a
monochromator then llight will
pass through the sample
solution placed in a cuvette
and its wavelength will be in
the digital display
Types of cuvette for UV
light
1. Glass cuvette
2. Plastic cuvette
3. Fused quartz
Types of cuvette for visible
light
1. Glass cuvette

Types of Spectrophotometers
Simple spectrophotometer
- Visible light
11. 380 to 750 nm
12. Light is produced by a
tungsten lamp
UV-vis spectrophotometer
13. Second
lamp
is
used:
DEUTERIUM turns a visible
light spectrophotometer into a
UV-visible unit that can
measure from: 190-380 nm
14. Available with a variety of
features: scanning, multiple
cells, intergral printers, and
user interfaces

ABSORBANCE AND
CONCENTRATION OF
ABSORBING MOLECULE
Absorbance vs Transmittance
1. Absorbance: The amount
of light that can be
absorbed
2. Transmittance:
The
amount of light that passes

through the solution as it is

not absorbed
3. Absorbance rather than
the transmittance is most
useful
in
spectrophotometry
4. If no light is absorbed
Absorbance = 0
Transmittance = 100%
5. Each unit in absorbance
corresponds with an order
of magnitude in the
fraction of light transmitted
A=1, 10% of the light is
transmitted (0.10) 90% is
absorbed
A=2, 1% of the light is
transmitted and 99% is
absorbed
A=3, 0,1% of light is
transmitted and 99,9% is
absorbed
Formula:
Transmittance, T = P / P0
% Transmittance, %T = 100 T
Absorbance,
A = log10 P0 / P
A = log10 1 / T
A = log10 100 / %T
A = 2 - log10 %T
What is the relation of
Concentration
and
Absorbance?
6. Explained by the Beers
Law
Beers Law
A=ebc

Where A is absorbance (no units,

since A
=
log10 P0 /
P)
e is the molar absorbtivity with units
of
L
mol-1 cm-1
b is the path length of the sample - that is, the path
length of the cuvette in which the sample is contained.
We will express this measurement in centimetres.

The vortex was used to

mix ALL the components
of the solution!
c. What is the accurate
absorbance
of
Bromophenol blue?

c is the concentration of the compound in solution,

expressed in mol L-

7. An
increase
in
the
absorbance will also lead
to an increase in the
concentration
(direct
relationship)
ACCURACY VS PRECISION
Linear Regression
y=mx + b
r= Pearsons correlation
coefficient/correlation coefficient
r= value should be near 1 or 1 for
the graph to be a linear graph

Accuracy
- How close the measured
value is to the actual value
or true value
a. Why was Bromophenol
blue (BPB) used in the
experiment?
8. BPB was used in the
experiment as an indicator
9. Can be used as a pH
indicator, color marker or
dye
b. Why was vortex used?

Precision
- How close the value of the
measurement is close to
the other measured values
by the group
Why is the analytical balance
accurate?

1. The analytical balance is

accurate
because
the
density of water is 1.0

C1V1=C2V2

!
Bromophenol blue: 1.25g/mL

Analytical Balance
EXPERIMENT 2
CELL STAINING TECHNIQUES
CELL
Appear transparent in in
vivo (under the microscope)
Staining the cells will show
specific cellular structures of
the cell
ex: Using the iodine solution
to stain the nucleus of the
Allium cepa
under the
brightfield microscope

Great
precision
in
quantitative
chemical
analysis
Can give up to 4 decimal
places (0.0000g)
Water: 1:1 ratio with mL
to g
DENSITY of water:1

Tetrahymena sp. (or hay infusion

in the experiment)
Single-celled
protozoan
(Protista)
Motile: Have cilia that is
used for cellular movement
Commonly
found
in
freshwater ponds

!
Formulas used:

!
Computing for Concentration (c2)

CELL STAINING
Stains used:
Lugols iodine, IKI (.15g.mL
dH2O)

Methyl green (.01 g/mL, 1%

HAc)
Nigrosin (.01g/mL dH20)

3. Nigrosin

1. Lugols Iodine
It is a solution of elemental
iodine and potassium iodide in
water. It is a mordant in gram
staining.
Neurtral stain
It will always change color
Staining:

Stains polysaccharides in
nucleus, vacuoles and cilia.

cell

Structures that are stained:

1. Nucleus
2. Glycogen vacuoles
3. Locomotory
organelles
(cilia)
2. Methyl Green
Methyl green is a basic dye
primarily
consisting
of
triphenylmethane.
Staining:

The structure of methyl green has

two positive charges, therefore
making it a cation. Since DNA is
negatively charged, brought about by
the presence of the phosphate group,
the cation, which is the dye, will bind
to the DNA (the anion).
Structures that are stained:
1. Nucleus (specifically
the micronuclei and
macronuclei)

An acidic dye made from the

oxidation of aniline, which is
an
organic
compound
consisting of phenyl group
attached to an amino acid.
Aniline is the prototypical
aromatic
amine.
Uses
negative staining technique.

Physical Form:
Black crystals or powder that is
soluble in water and slightly soluble
in ethanol
Acidic Dye:
Easily/readily gives up a hydrogen
ion and becomes negatively charged

Negative Staining:
Instead of staining the cell, the dye
stains the background thus making
the outline of the cell surface visible
Structures that are stained:

Cell surface or overall

structure of the cell

Lugols Iodine
Methyl Green
Nigrosin

Structures
Stained
Nucleus,
glycogen
vacuoles, cilia
Nucleus
Cell sruface

EXPERIMENT 3
PHAGOCYTOSIS IN
TETRAHYMENA sp.
Phagocytosis in a Tetrahymena
(black: food vacuoles)

Parts (in color)

Oral apparatus or cytosome
Food vacuole
Contractile vacuole
Macronucleus
Micronucleus
Longitudunal microtubule bands

Many food particles cannot

easily pass through the cellmembrane of a single-celled
organisms
through
diffusion !
FOOD PARTICLES SHOULD
BE INGESTED by the cells
through ENDOCYTOSIS !
Endocytosis!

A process in which the cell takes in

materials from the outside by
ENGULFING
FUSING the food particles
with its plasma membrane !
Phagocytosis!
A type of endocytosis
which
is
CELL
ENGULFING

Cells ingest large food

particles (or smaller
cells) !
Pinocytosis !
A type of endocytosis
which
is
CELL
DRINKING
Involves very minute
food particles or liquid
substances !

Food particles used

1. Graphite shavings from
pencil (insoluble)
2. India ink, 1% (soluble) !
How does the Tetrahymena take in
food?
The Tetrahymena makes
use of its CILIA in order to
create currents that will
move the food particles to
its oral cavity
Food will be ingested from
its oral cavity !
CILIA near its oral cavity is
specialized for the FEEDING
of the Tetrahymena !
Right side of the oral cavity:
Feeding structure called the
UNDULATING MEMBRANE
(made of cilia)
Left side of the oral cavity:
Three smaller ciliary
membranellae that curve
clockwise toward the
cytosome
Food particles will move
from the oral cavity to the
cytosome
PHAGOCYTOSIS:
o When food reaches the
cytostome - it is
enclosed
in
a
membrane
bound
food vacuole that

pinches
off
and
migrates through the
cell.
Graphite shavings
2. Insoluble in water
3. Phagocytosis
India Ink

Soluble in water
Pinocytosis
An increase of concentration
in india ink, less food
vacuoles

Formalin
Used for fixation
Formaldehyde

EXPERIMENT 4
CELL COUNTING USING
THE DYE EXCLUSION
PRINCIPLE
1. The DYE EXCLUSION TEST
- Used to determine
the number
of
viable cells present
in a cell suspension
- PRINCIPLE
Live cells have an
intact
cellular
membrane which
can
actively
exclude
certain
dyes
1. Typan blue
2. Eosin
3.Propidium
idodide
Dead cells do not
have
such
integrity and are
stained BLUE

Consits of TWO chambers

One chamber is divided into 9
large
squares
with
the
dimension of 1x1mm
A cover glass (0.1mm) is
supported over these squares
TOTAL VOLUME OF EACH
SQUARE
1.00mmx0.1mm or
0.1mm^3 or
10^-4cm^3
EACH LARGE SQUARE
HAS
A
VOLUME
OF
0.0001mL

2. Hematocytometer is used in
cell counting
HEMATOCYTOMETER

3.
4.
5.

Counting using a hemocytometer

Cell count in a hemocytometer
depends on:
Even distribution of cells in
suspension

Improper
filling
of
chambers (too much or too
little)
Representative
smaple
taken for counting with the
pipette and added with no
air bubbles
Counting cells that are
attached to each other or
in
contact
with
the
enclosing boundaries
The total number of
chambers counted within
the hemocytometer
The number of cells
counted

Bacteria used: Saccharomyces

cerevisiae (cell suspension)
Bacteria used in yeast
Baking, alcohol
Counting the cells using the
hemacytometer
Clean the hematocymeter and
cover slip with 70% ethanol
Use a brightfield microscope
The bacteria must be mixed
with 0.3% Trypan blue in 3%
formalin
(trypan
blue,
staining the dead cells blue
and formalin is for the
fixation of the cell)
Only introduce the mixture
into one side of the cell
chamber (2 chambers) and
make sure the mixture covers
the ENTIRE GRID IN ONE
CONTINUOUS
MOTION
WITH NO BUBBLES AND
FLUID
DOES
NO
OVERFLOW
INTO
THE
GUTTER
Viable cells are transparent
Nonviable cells are color
blue

Only count the cells within

the squares including those
that touch the midline at the
top and left
If there are more than 50
cells per square, DILUTE
the cell suspension with the
twice amount of PBS
(Phosphate Buffer Saline)
originally used
Phosphate Buffer Saline
pH is 7.4
Balanced salt solution
Used for diluting cells
Can also be used for washing
cells
before
dissociation,
transporting cells or tissues or
preparing reagents
No calcium, magnesium or
phenol red

ORDINARY
GLASSS
COVERSLIPS CANNOT be
used
with
the
hemacytometer

glass
coverslip used:
Using the wrong coverslip
can:
1. Distort the cell due to the
capillary force of the liquid
under the coverslip unless it is
measured to the precise
amount to fill the space under
the coverslip
2. Lead to an inaccurate count of
cells

USE NEUBAUER
IMPROVED hematocymeter

Spectrophotometry and Cell

counting

Determining the Cell Density

OPTICAL DENSITY
Optical density is applicable to
all solutions: CELL
SUSPENSION
Spectrophotmeter 600nm

EXPERIMENT 5
RESTRICTION ENDONUCLEASE
DIGESTION OF PLASMID DNA
What
are
endonucleases?

Reagent blank: NO
SUPERNATANT (no cell
suspension in the medium
EQUATIONS

%viability
=
#viable
cells/
(#nonviable+#viable cells) x100
Cells/mL = average count of cells
x dilution factor x 10^4
Dilution Factor = cell suspension
+ Trypan Blue + Phosphate Buffer
Saline / cell suspension volume
#doublings
=
concentration 1)
concentration 2)/ log 2

log(cell
log (cell

growth rate = #doublings/time

doubling time = 1/growth rate

restriction

ENZYMES that cleave the

sugar-phosphate backbone
of DNA at specific sites
Only cleave at specific sites
In several cases, a given
enzyme cuts both strands
ofduplex DNA within a stretch
of just a few bases
FROM BACTERIA, a large
majority
of
restriction
enzymes have been isolated
and appear to serve a hostdefense role
Foreign
DNA
(from
an
infected virus) will be chopped
up and inactivated (restricted)
within the bacterium by the
restriction enzymes
Hamilton O. Smith and Kent
Wilcox were the first to
characterize a type II
restriction enzyme, HindII,
which led to the further
development
of
the
recombinant
DNA
technology.

How are restriction enzymes

named?
After their HOST OF ORIGIN
Example:
1. EcoRI
Isolated from: Escherichia coli
(strain RY13)
2. HindIII
Isolated from Haemophilus
influenzae
3. BamHI

Isolated from: Bacillus

amyloliquefaciens

Restriction
enzymes
hydrolyze the backbone of
DNA between deoxyribose
and phosphate group
This will leave a phosphate
group on the 5 ends and a
hydroxyl group on the 3
ends of both strands
A few restriction enzymes
will cleave single stranded
DNA although at LOW
efficiency

Types of Recognition Site

Ends
5 overhangs
an asymmetric cut that
produces a shorter 5 end

3 overhangs
similar to a 5 overhang but
the shorter end is observes in
the two 3 ends.

Blunt End
symmetric
cuts
that
produce
equal
length
strands.

pBR322 DNA

Restriction
endonuclease
combination:
ScaI + Sal I
2 Fragments
Lengths:
Fragment 1 = 1, 168bp
Fragment 2 = 3, 193bp

3. Restriction encoduclease
combination: PstI + BamHI
+ ScaI
3 Fragments
Lengths:
-ScaI-BamHI: 892 bp
-

1.
2.

Artificially created plasmid

Circular, double stranded
DNA
4361 base pairs
Used as an E. coli cloning
vector
Restriction
endonuclease
combination:
BamHI + EcoRI
2 Fragments
Lengths:
Fragment 1 = 377bp
Fragment 2 = 3, 984bp

BamHI-PstI: 3, 232 bp
-PstI-ScaI: 237 bp

4. Restriction
endocuclease
combination: Eco RI + Sal I
+ Pst I
3 Fragments
Lengths:
Fragment 1: 653
bp
Fragment 2: 2, 956
bp
Fragment 3: 752
bp

When the recognition site

for each of the restriction
enzymes
used
in
the
experiment is known, the
base pair sequence in a
small region at which the
pBR322 DNA is cleaved is
therefore determined. This
information can be used to
study specific regions in
the pBR322 DNA.
Notes:
In Restriction Digestion, buffer
then Restriction Enzyme then
DNA
Each Restriction Enzyme has
its
own
APPROPRIATE
BUFFER
Restriction enzymes need to
be placed first before the
buffer as these enzymes work
fast when it comes to splicing
the DNA sequence
AGAROSE GEL
ELECTROPHORESIS

5. Restriction
endonuclease
enzyme: Bam HI + Sal I +
6.
7. Sca I
3 Fragments
Lengths:
Sca I - Bam HI: 892
bp
Bam HI - Sal I: 276
bp
Sal I - Sca I: 3,193
bp

1g of Agarose powder in
100mL 1xTAE buffer
1% AGAROSE GEL
Agarose gels are commonly
used in concentrations of
0.7% to 2% depending on the
size of bands needed to be
separated

TAE BUFFER
Buffer solution containing a
mixture of Tris base, acetic
acid and EDTA. I
n molecular biology it is used
in agarose electrophoresis
typically for the separation
of nucleic acids such as

from each other, and a lower

percentage gel will help separate
larger bands.
DNA LADDER
Used to determine the DNA
bands (DNA band readings
are compared to the DNA
Ladder)

DNA and RNA. It is made up

of Tris-acetate buffer, usually
at pH 8.0, and EDTA, which
sequesters divalent cations.

Why is Ethidium bromide used?

Intercalating agent
Fluorescent tag (Nucleic
acid stain)
How do you get better resolution
of bands?
a) running the gel at a lower voltage
for a longer period of time
b) using a wider gel comb
c) loading less DNA into well
How do you get better separation
of bands?
If you have similarly sized bands that
are running too close together you
can adjust the agarose percentage
of the gel to get better separation.
A higher percentage agarose gel
will help resolve smaller bands

GEL
ELECTROPHORESIS
RESULTS
CONCENTRATION
can
affect the mobility of the
DNA and may cause it to be
more
condensed
and
aggregating thus restricting
the movement of DNA
The band will be compared
with the DNA Ladder
EXPERIMENT 6
Extracting DNA from E.coli
- DNA Extraction is the
process of isolating DNA
from cells by mechanical
and
biochemical
methods
- DNA extracted will be
used
to
carryout
a
downstream of genetic
experiments
or
DNA
analyses
- DNA extraction is a major
procedure in molecular
biology and forensic
science and an essential
technique
used
by
biological and medical
scientists
DNA Extraction
1.
Cells containing DNA
will be opened
chemically or

2.

3.

mechanically to release
DNA
DNA is in a suspension,
it will be purified from
contaminating cellular
components such as
lipids in the cell
membrane by
washing it with SDS
or a detergent
DNA is precipitated
using cold alcohol and
resuspended in an
appropriate volume

protocol according to their

laboratory equipment,
reagents and target
samples

The purity of DNA is tested

using
the
spectrophotometer
by
measuring
the
concentration of DNA
The
wavelength
of
nucleic acid is 260 nm
while proteins have 280
nm

EDTA
Ethanol (70%)
SDS (Sodium dodecyl
sulfate, 20%)
NaCl
TE buffer
EXPERIMENT 7
Marker Gene Amplification Using
Polymerase Chain Reaction
- Replication in a tube
- Discovered by Kary
Mullis
- Fundamental laboratory
equipment of molecular
biology
- Amplify a particular DNA
SEQUENCE to extremely
high copy numbers
- Researchers may
comfigure their PCR

What is needed in PCR master

mix?
Primers are short pieces of DNA
that are made in a laboratory. Since
they're custom built, primers can
have any sequence of nucleotides
you'd like

DNA Polymerase is a naturally

occurring complex of proteins whose
function is to copy a cell's DNA
before it divides in two. When a DNA
polymerase molecule bumps into a
primer that's base-paired with a
longer piece of DNA, it attaches itself
near the end of the primer and starts
adding nucleotides. (In nature, these
primers are made by an enzyme
called primase).
PCR buffer
Provides a suitable chemical
environment for optimum activity
and
stability
of
the
DNA
polymerase
Taq DNA polymerase
Almost all PCR applications employ
a heat-stable DNA polymerase, such
as Taq
polymerase (an
enzyme
originally
isolated
from
the
bacterium Thermus aquaticus). This
polymerase enzymatically assemble
s a new DNA strand from DNA
building-blocks, the nucleotides, by
using single-stranded DNA as a
template
and
DNAoligonucleotides (also
called DNA primers), which are
required for initiation of DNA
synthesis.
The Taq polymerase optimum
temperature is 70 C.
dNTP
mix
(Deoxynucleotides
triphosphate mix)
The building-blocks from which the
DNA polymerase synthesizes a new
DNA strand
MgCl2
Generally Mg2+ is used, but Mn2+ can
be
used
for
PCR-mediated
DNAmutagenesis,
as
higher

Mn2+ concentration increases the

error rate during DNA synthesis[9]
Sterile H2O
PCR and Thermocycling
The method relies on thermal
cycling, consisting of cycles of
repeated heating and cooling of the
reaction
for DNA
melting and enzymatic replication of
the DNA.
The cycles in PCR means that it is
the temperature changes occurring
Steps In PCR
1. Initialization
This step consists of heating the
reaction to a temperature of 94
96 C (or 98 C if extremely
thermostable polymerases are used),
which is held for 19 minutes.
2. Denaturation
This step is the first regular cycling
event and consists of heating the
reaction to 9498 C for 2030
seconds. It causes DNA melting of
the DNA template by disrupting the
hydrogen
bonds
between
complementary bases, yielding
single-stranded DNA molecules.
3. Annealing
The reaction temperature is lowered
to 5065 C for 2040 seconds
allowing annealing of the primers to
the single-stranded DNA template.
This temperature must be low
enough to allow for hybridization of
the primer to the strand, but high
enough for the hybridization to be
specific, i.e., the primer should only
bind to a perfectly complementary
part of the template.

The primers begin to

attach to the DNA
template

4. Extension/Elongation
The temperature at this step
depends on the DNA polymerase
used; Taq
polymerase has
its
optimum activity temperature at 75
80 C,[12][13] and
commonly
a
temperature of 72 C is used with
this enzyme.
At this step the DNA polymerase
synthesizes a new DNA strand
complementary to the DNA
template strand by adding dNTPs
that are complementary to the
template in 5' to 3' direction,
condensing
the
5'-phosphate
group of the dNTPs with the 3'hydroxyl group at the end of the
nascent (extending) DNA strand.
The extension time depends both on
the DNA polymerase used and on
the length of the DNA fragment to
amplify.
5. Final elongation
This single step is occasionally
performed at a temperature of 70
74 C (this is the temperature
needed for optimal activity for most
polymerases used in PCR) for 515
minutes after the last PCR cycle to
ensure that any remaining singlestranded DNA is fully extended.
6. Final hold
This step at 415 C for an indefinite
time may be employed for short-term
storage of the reaction.

EXPERIMENT 8
Extraction and Amplification of
Rice Genomic DNA
- Rice, Oryza sativa has
several varieties such as
C-4 and IR-64
- Sinandomeng, Jasmine,
Black, Risotto, Basmati

Different types of rice can

be identified by molecular
means through marker
genes such as the
RG100
The
amplification
process is just the same
as in experiment 7

EXPERIMENT 9
Bioinformatics tools for Cell and
Molecular Biology
- Bioinformatics is the
mathematical, statistical
and computing methods
tool
used
to
solve
biological problems using
DNA and amino acid
sequence and related
information
- The
goal
of
bioinformatics
is
to
uncover the wealth of
biological
information
hidden in the mass of
data and obtain a clearer

insight
into
the
fundamental biology of
organisms
The
application
of
computer technology to
the
management
of
biological
information.
Computers are used to
gather, store, analyze and
integrate biological and
genetic information which
can then be applied to
gene-based
drug
discovery
and
development.

Application/Software utilized
MEGA
(Molecular
Evolutionary
Genetics)
- Application since 1993
- MEGA6
- Uses DNA sequence,
protein
sequence,
evolutionary distance or
phylogenetic tree data