S.Y.
BSC SEMESTER III
BOTANY PAPER II
UNIT I: INSTRUMENTATION & TECHNIQUES
CHROMATOGRAPHY
BY Miss. Sonam Shukla
Department of Botany
Satish Pradhan Dnyanasadhana College, Thane (w)
Agarose gel electrophoresis is a method to
separate DNA, or RNA molecules by size.
This is achieved by moving negatively charged
nucleic acid molecules through an agarose
matrix with an electric field (electrophoresis).
Shorter molecules move faster and migrate
farther than longer ones .
At any given PH, exist in a solution as
electrically charged species either as a cation
(+) or anion(-).
Under the influence of an electric field these
charged particles will migrate either to cathode
or anode, depending on the nature of their net
charged
Electrophoresis is the movement of molecules
by an electric current.
Nucleic acid moves from a negative to a
positive pole.
• DNA is negatively charged.
• When placed in an electrical field, DNA will migrate
toward the positive pole (anode).
• An agarose gel is used to slow the movement of DNA
and separate by size.
H O2
DNA
- +
Power
Power supply and chamber, a source of negatively
charged particles with a cathode and anode
Buffer, a fluid mixture of water and ions
Agarose gel, a porous material that DNA migrates
through
Gel casting materials
DNA ladder, mixture of DNA fragments of known
lengths
Loading dye, contains a dense material and allows
visualization of DNA migration
DNA Stain, allows visualizations of DNA fragments
after electrophoresis
Electrophoresis Equipment
Power supply
Gel tank Cover
Electrical leads
Casting tray
Gel combs
Agarose Gel
A porous material derived from
red seaweed
Acts as a sieve for separating DNA
fragments; smaller fragments
1% agarose
travel faster than large fragments
Concentration affects DNA
migration
Low conc. = larger pores
better resolution of larger DNA 2% agarose
fragments
High conc. = smaller pores better resolution
of smaller DNA fragments
Agarose Buffer Solution
Combine the agarose powder and buffer solution. Use a flask that is
several times larger than the volume of buffer.
Melting the Agarose
Agarose is insoluble at room temperature (left).
The agarose solution is boiled until clear (right).
Pouring the gel
DNA
buffer
wells
Anode
Cathode (positive)
(negative)
Sample Preparation
Loading the Gel
Running the Gel
Electrophoresis Buffer
TAE (Tris-acetate-EDTA) and TBE (Tris-borate-
EDTA) are the most common buffers for duplex DNA
Establish pH and provide ions to support conductivity
Concentration affects DNA migration
Use of water will produce no migraton
High buffer conc. could melt the agarose gel
New Sodium Borate (SB) buffer allows gels to be
run at higher voltages in less time than traditional
buffers
Loading Dye
DNA samples are loaded into a gel AFTER the
tank has been filled with buffer, covering the gel
Contains a dense substance,
such as glycerol, to allow the
sample to "fall" into the sample
wells
Contains one or two tracking
dyes, which migrate in the gel
and allow monitoring of how
far the electrophoresis has
proceeded.
DNA Staining
Allows DNA
visualization after gel
electrophoresis
Ethidium Bromide
In gel staining
Analysis
After electrophoresis the gel is illuminated
with an ultraviolet lamp to view the DNA
bands. The ethidium bromide fluoresces
reddish-orange in the presence of DNA.
photograph it with a digital camera.
Applications
Estimation of the size of DNA molecules following
restriction enzyme digestion, e.g. in restriction
mapping of cloned DNA.
Analysis of PCR products, e.g. in molecular genetic
diagnosis or genetic fingerprinting
Separation of DNA fragments for extraction and
purification.
Separation of restricted genomic DNA prior to
Southern transfer, or of RNA prior to Northern
transfer.
VERTICAL
GEL ELECTROPHORESIS
Experimental Goals
To understand the principle of SDS-PAGE
To become familiar with the SDS-PAGE
setup
What is Electrophoresis?
Electrophoresis is a
laboratory technique for
separating molecules
based on their charge
The gel (matrix)
The gel (matrix) itself is composed of either
agarose or polyacrylamide.
Polyacrylamide is a cross-linked polymer of
acrylamide.
Acrylamide is a potent neurotoxin and should be
handled with care!
Polyacrylamide gels
Have smaller pores than agarose, therefore high
degree of resolving power.
Can separate DNA fragments which range in
size from 10-500 bp.
DNA fragments which differ in size by one
nucleotide can be separated from each other.
Polyacrylamide gel electrophoresis is also used
to separate protein molecules.
Protein Electrophoresis
Separate proteins based on
Size (Molecular Weight - MW)
Allows us to
characterize
quantify
determine purity of sample
compare proteins from different
sources
Protein Electrophoresis
Proteins, unlike DNA, do not have a
constant size to charge ratio
In an electric field, some will move to the
positive and some to the negative pole, and
some will not move because they are neutral
Native proteins may be put into gel systems
and electrophoresed
An alternative to native protein gels forces
all proteins to acquire the same size to
charge ratio
SDS-PAGE
SDS-PAGE ( sodium dodecylsulphate-
polyacrylamide gel electrophoresis)
The purpose of this method is to separate
proteins according to their size, and no other
physical feature
In order to understand how this works, we
have to understand the two halves of the name:
SDS and PAGE
Sodium Dodecylsulphate
SDS (sodium dodecyl sulfate) is a
detergent that can dissolve hydrophobic
molecules but also has a negative charge
(sulfate) attached to it.
If SDS is added to proteins, they will be
soluablized by the detergent, plus all the
proteins will be covered with many
negative charges.
Polyacrylamide Gel Electrophoresis (PAGE)
PAGE is the preferred method for separation of
proteins
Gel prepared immediately before use by polymerization
of acrylamide and N,N'-methylene bis acrylamide.
Porosity controlled by proportions of the two
components.
Catalyst of polymerization
Polymerization of acrylamide is initiated
by the addition of ammonium persulphate
and the base N,N,N’,N’-
tetramethylethylenediamine (TEMED)
TEMED catalyses the decomposition of
the persulphate ion to give a free radical
Polyacrylamide Gels
Bis-Acrylamide polymerizes along with acrylamide
forming cross-links between acrylamide chains
Polyacrylamide Gels
Pore size in gels can be varied by varying the ratio of
acrylamide to bis-acrylamide
Protein separations typically use a 29:1 or
37.5:1 acrylamide to bis ratio
Side view
Movement of Proteins in Gel
Movement of Proteins in Gel
smaller proteins
will move through
the gel faster while
larger proteins
move at a slower
pace
Components of the System
DC Power Source, Reservoir/Tank, Glass
Plates, Spacers, and Combs
Support medium
Gel (Polyacrylamide)
Buffer System
High Buffer Capacity
Molecules to be separated
Proteins
Nucleic Acids
Vertical Gel Format:
Polyacrylamide Gel Electrophoresis
Reservoir/Tank
Power Supply
Glass Plates, Spacers, and Combs
Step by Step Instructions on how to assemble the
polyacrylamide gel apparatus
Prepare polyacrylamide gels
Add diluted samples to the sample buffer
Heat to 95C for 4 minutes
Load the samples onto polyacrylamide gel
Run at 200 volts for 30-40 minutes
Stain
Reagent 8% (Running Gel) 5% (Stacking Gel)
Acrylamide/ Bisacrylamide 4.0 mls 2.5 mls
(40%) *
1 M Tris-HCl pH 8.8 7.5 mls 7.5 mls
water (distilled) 8.2 mls 9.7 mls
10% SDS 200 µl 200 µl
10% Ammonium Persulfate 100 µl 100 µl
TEMED (added last) 10 µl 10 µl
* = 19:1 w:w ratio of acrylamide to N,N'-methylene bis-acrylamide
Mix ingredients GENTLY! in the order shown
above, ensuring no air bubbles form.
Pour into glass plate assembly CAREFULLY.
Overlay gel with isopropanol to ensure a flat
surface and to exclude air.
Wash off isopropanol with water after gel has set
(+15 min).
Tris buffer to provide appropriate pH
SDS (sodium dodecyl sulphate) detergent to
dissolve proteins and give them a negative charge
Glycerol to make samples sink into wells
Bromophenol Blue dye to visualize samples
Heat to 95C for 4 minutes
Loading Samples & Running the gel
Run at 200 volts for 30-40 minutes
Running Buffer, pH 8.3
Tris Base 12.0 g
Glycine 57.6 g
SDS 4.0 g
distilled water to 4 liter
• Coomassie Brilliant Blue
•The CBB staining can detect about 1
µg of protein in a normal band.
• Silver Staining
•The silver stain system are about 100
times more sensitive, detecting about
10 ng of the protein.
Protein bands observed in SDS-PAGE
Coomassie Brilliant Blue
Molecul
ar
Weight
Standar
250
KD
d
150
100
75
50
37
25
20
15
10
Example of silver stained gel
Silver staining is usually
10-100 times more
sensitive than
Coomassie Blue
staining, but it is more
complicated.
Faint but still visible
bands on this gel contain
less than 0.5 ng of
protein!
