Gel Electrophoresis ......................

gel electrophoresis

1. BIOL 2100
GENERAL BIOTECHNOLOGY
KHRISTINA JUDAN CRUZ, PhD
Department of Biological Sciences
College of Science
Central Luzon State University
Detection of extraction and
amplification products

2. □Gel electrophoresis
□Spectrophotometry
Methods in detecting of
extraction and amplification
products

3. Electrophoresis
□a technique commonly used in the lab
to separate charged molecules
according to size.

4. □Charged molecules move through a
gel when an electric current is passed
across it
□An electric current is applied across
the gel so that one end of the gel has
a positive charge and the other end
has a negative charge

5. □The movement of charged molecules
is called migration
□The gel consists of a permeable
matrix through which molecules can
travel when an electric current is
passed across it

6. Fractionating DNA Molecules
DNA is negatively charged at
neutral pH
DNA will travel towards a
positive charge
DNA electrophoresed in a gel
will travel according to its
chain length

7. Gel Electrophoresis of DNA
□Gel electrophoresis detects the presence of DNA
in a sample
□Gel electrophoresis detects the number of
nucleotides in a fragment of DNA
■e.g., the number of nucleotides in a DNA
region which was amplified by PCR
■Is a rough estimate, is not exact, need more
sophisticated sequencing techniques to get an
exact number of nucleotides
■Can be used to tentatively identify a gene
because we know the number of nucleotides in
many genes

8. How Gel Electrophoresis of DNA
Works
□A sample which contains fragments of DNA is forced
by an electrical current through a firm gel which is
really a sieve with small holes of a fixed size
■Phosphate group in DNA is negatively charged so it is
moved towards a positive electrode by the current
■Longer fragments have more nucleotides
□So have a larger molecular weight
□So are bigger in size
□So aren’t able to pass through the small holes in the
gel and get hung up at the beginning of the gel
■Shorter fragments are able to pass through and
move farther along the gel
■Fragments of intermediate length travel to about the
middle of the gel
□DNA fragments are then visualized in the gel with a
special dye
□The number of nucleotides are then estimated by
comparing it to a known sample of DNA fragments
which is run through the gel at the same time

9. How Gel Electrophoresis of DNA
Works
□Reagents Needed
■Sample of DNA fragments
■Known sample of DNA fragments
□DNA ladder
■Gel
□Agarose
■Dye to visualize the movement of the sample as it
is traveling through the gel
□Loading dye – Blue juice
□So know when to stop so sample doesn’t just
run out of the gel
■Dye to visualize DNA after it has traveled to its
final spot in the gel
□Syber® Safe
■Buffer

10. Gel Electrophoresis
□Equipment Needed
■Box to hold the gel
■Comb to create small wells in the agarose
gel to put the DNA sample into at the
beginning of the gel
■Positive and negative electrodes to create
the electrical current
■Power supply
■Gel photo imaging system

11. A gel sits within a
tank of buffer. The
DNA samples are
placed in wells at one
end of the gel and an
electrical current
passed across the
gel. The negatively-
charged DNA moves
towards the positive
electrode.
Image credit:
Genome Research
Limited

15. How Gel Electrophoresis of DNA
Works

16. Dr.Saba Abdi
Agarose Gel
Stained with ethidium bromide (EtBR) to Visualize the DNA
Screening
PCR products
to test for the
presence of
specific DNA
sequences
500 bp
molecular
weight
markers
molecular
weight
markers
correct
PCR
product
600 bp
700 bp
1000 bp
slots where
DNA is loaded
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17. If properly done, genomic extraction should result in bright bands in
the very high base pair range of a gel electrophoresis.
Sizes of Genomic DNA for
various Species in kbp
E. Coli 4,640,000bp
Yeast 12,100,000bp
Fruit Fly 140,000,000bp
Human 3,000,000,000bp
Pea 4,800,000,000bp
Wheat 17,000,000,000bp
Analyzing DNA Samples

18. □gel box
□used to separate
DNA in an agarose
gel with an
electrical charge
□Different sized
pieces of DNA
move at different
rates

19. Spectrophotometry
□the quantitative measurement of the
reflection or transmission properties of
a material as a function of wavelength
(nist.gov)

22. □a method to measure how much a
chemical substance absorbs light by
measuring the intensity of light as a
beam of light passes through sample
solution
□The basic principle is that each
compound absorbs or transmits light
over a certain range of wavelength

23. □the spectrophotometer measures
quantitatively the amount of light
passing through a compound in
solution as a fraction of the light
emitted by the machine

25. Dr.Saba Abdi
Using Spectroscopy to analyze DNA
DNA absorbs UV light with a major peak at 260 nm
Optical Density
Wave Length
260
This absorption is useful because
it varies with the structure of
DNA (&RNA)
i.e. absorption coefficient
depends on the structure
dsDNA
Low
absorption
coefficient
ssDNA
Higher
absorption
coefficient
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26. Dr.Saba Abdi
Evaluation of Nucleic Acids
A2601.0 ≈ 50 µg/mlDNAA260/A2801.6 - 1.8A2601.0 ≈ 40 µg/mlRNAA260/A280~2.0
•spectrophotometrically
•quantity
•quality
•fluorescent dyes
•gel electrophoresis
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