Practical section of lab. method ppt 2017 2018

Practical Course of
Laboratory Methods
2017-2018

Laboratory methods description
• Laboratories use a variety of methodologies to test the
countless analytes that are of interest to the agriculture
community. Understanding the method used for a test provides
a broader context for understanding your test results
Team of work: 2017-2018
Prof. Dr. Ahmed H. Abo Dodoma Prof. Dr. Khaled Fahmy
Assistant Instructor: Mona M. Moghazee
Assistant Instructor: Hala Zoghly
Instructor: Hader Yousry

Preparation
of solution
Lab. methods
Level 3
2017-2018

Outlines:
• Objective
• Case study
• Types of chemical materials.
• Real solution vs. buffer solution.
• Solution Preparation Forms
• Laboratory tasks
• Dilution

Objective
preparation of solution
• You will be able to make a solution
from the solid and dilute it to a
specific concentration.

Case study
5 M NaCl
• Nucleic acid
extraction
1% agarose
gel
• Agarose gel
preparation
PCR mix
preparation
• dNTPs
• Primers
• Taq polymerase

Types of Materials
Solution Powder
What’s different between
Buffer & Solution?

Solution
• Solution:
• A uniform
homogeneous
mixture of two or
more substances. The
individual substances
may be present in
varying amounts.

Solution component:
Solute: The substance which is dissolved,
or has gone into solution (typically a
solid).
Solvent: The substance which does
the dissolving (typically a liquid,
such as water or alcohol). Must
be greater than 50% of the
solution.

Buffer
• Buffer:
• A solution which tends
to maintain a constant
pH when excess acid
or base is added.

Solution Preparation Forms
Percentages %= 5% SDS
5g of SDS up to 100ml
Molar concentrations (M)=
g= Mw x Mx L
Dilution= C1 V1 =C2 V2

Laboratory Tasks
✓Prepare a solution of 1.2%
NaCl (%w/v)
✓Prepare 40 ml of 1.2% NaCl
✓Prepare 1000 ml of 1M NaCl

1. Dilution by Water
•Cb x Vb = Ca x Va
• Where C = concentration, V = volume,
• b = before dilution and a = after dilution
• Example:
If we add 20 ml of water to 30 ml of 2% NaCl the final volume
will be 50 ml and the final concentration will be 1.2%
Cb x Vb = Ca? x Va
Ca = Cb x Vb / Va
Ca = 2 x 30 / 50 = 1.2%

2. Dilution By mixing different
concentrations
• Cf x Vf = (C1 x V1) + (C2 x V2)
• f = final solution, 1 = 1st solution and 2 = 2nd solution
• Example: if we add 20 ml of 1% NaCl to 30 ml of 2% NaCl the
final volume will be 50 ml and the final concentration will be
1.6%
• Cf? x Vf = C1 x V1 + C2 x V2
• Cf = [C1 x V1 + C2 x V2] / Vf
• Cf = [1 x 20 + 2 x 30] / 50 = 1.6%

3. Dilution By mixing different
solutions
• Cb x Vb = Ca x Va
• b = before mixing and a = after mixing
• Example: if we add 20 ml of 1% KCl to 30 ml of 2% NaCl the final
volume will be 50 ml and the final concentration of NaCl will be 1.2%
and for KCl will be 0.4%
Cb x Vb = Ca x Va
Ca = Cb x Vb / Va
Ca [for NaCl] = 2 x 30 / 50 = 1.2%
Ca [for KCl] = 1 x 20 / 50 = 0.4%

Laboratory Tasks
✓Prepare a solution of 1.2%
NaCl (%w/v)
✓Prepare 40 ml of 1.2% NaCl
✓Prepare 1000 ml of 1 M NaCl
✓Prepare 0.5 % NaCL from
1% NaCl solution

Researcher
(Arnold Beckman )
Chemist, Scientist, Educator (1900–2004)
• Arnold Beckman worked as a chemist and professor before
creating the "acidimeter," meant to measure acidity levels and
thus becoming the first pH meter. Beckman founded his own
business, Beckman Instruments, and invented a number of
additional items, including the Beckman Spectrophotometer.

Devices & laboratory
equipment

Explain, How the
Devices work and
precautions of
these devices that
use in preparation
of solution?
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10

Explain, How this
Device work and
the precautions of
this?
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Blotting technology Lab. Methods

Letter S1
Lab. methods
Level 3
2017-2018

Calculation
PictureDescriptionName
Tool
Researcher
Technology
/Technique
Device
-------/ 20-------/ 40-------/ 40Sumiton =
------/100Total=

Tools
“Syringe”
A tool used for injecting or withdrawing
liquids.

Explain technique that works on this tool?
• …………………………………
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10

Researcher
(Susumu Tonegawa)
• Famous As: Immunologist,
Molecular Biologist.
• In 1987, he was awarded the
Nobel Prize in Physiology or
Medicine "for his discovery of
the genetic principle for
generation of antibody
diversity."

Mention another researcher
begins with a letter S
• ………………………………………………………
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10

Technology or Technique
or Terms
“Southern blotting”

Markers in
biology:
(genetics)
A gene or DNA
sequence with known
physical location, and
whose pattern
of inheritance can be
followed.
Molecular markers
• Based on hybridization
• Based on PCR generation
Biochemical
markers
• Isozyme
• Protein banding pattern

Southernblotting
definition
• The southern blotting principle is
Hybridization, Which is the
process of forming a double
stranded DNA molecule
between a single-stranded DNA
probe and a single-stranded
target DNA.

Southern blotting steps
1. Extract and purify DNA from cells.
2. DNA is restricted with enzymes.
3. Separated by electrophoresis.
4. Denature DNA.
5. Transfer to nitrocellulose paper.
6. Add labeled probe for hybridization to take place.
7. Wash off unbound probe.
8. Autoradiograph.

Southern blotting
Advantages
• Effective way to detect a
specific DNA sequence in
large, complex sample of
DNA.
• Can be used to quantify the
amount of the present DNA.
• Cheaper than DNA
sequencing.
Dis-advantages
• More expensive than most
other tests.
• Complex and labor
intensive.

Southern blotting applications
To identify specific
DNA in a DNA
sample.
1
To Isolate desired
DNA for construction
of rDNA.
2
Identify mutations,
deletions, and gene
rearrangements.
3
In DNA
fingerprinting
4

In the light of your
previous study,
• what are the tools and
equipment needed to do
the southern blotting
technique?
• …………………………………………
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10

Device
Spectrophotometer
spectrophotometry is the
quantitative measurement of the
reflection or transmission properties
of a material as a function of
wavelength.
Spectrophotometry is an important
technique used in many biochemical
experiments that involve DNA, RNA,
and protein isolation, enzyme
kinetics and biochemical analyses.

Explain the technique that
works on this device?
• ………………………………………………………
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10

Letter N
Lab. methods
Level 3
2017-2018

Tools
“Needle of inculcation”
It is a laboratory
equipment used in the
field of microbiology to
transfer and inoculate
living microorganisms

Researcher
(Norman Borlaug)
• Famous As: Father Of The Green Revolution
• Nationality: America
• Over the course of his work he
successfully developed disease
resistant, high yielding wheat
varieties which when combined with
modern agricultural production
techniques could dramatically change
the way farming was done.

begins with a letter N
• ………………………………………………………
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Technology or Technique or
Terms
“Northern blotting”

Northernblotting
definition
• The northern blotting principle is
Hybridization, Which is the
process of forming a double
stranded DNA-RNA-hybrid
molecule between a single-
stranded RNA probe and a
single-stranded target RNA.

Northern blotting steps
1. Extract and purify mRNA from cells.
2. Separated by electrophoresis.
3. this gel is immersed in depurination buffer for 5-10 minute
then washed with water.
4. Transfer to aminobenzyloxymethyl filter paper.
5. After transfer, the membrane is baked at 80ᵒc
6. Add labeled probe for hybridization to take place.
7. Wash off unbound probe.
8. Autoradiograph.

Southern blotting
vs. Northern blotting
Southern blotting Northern blotting

Northern
blotting
applications
To detect specific
mRNA in a sample.
To screen
recombinants by
detecting the mRNA
produced by the
transgene
Diseases
diagnosis.
Gene expression
studies

previous study,
the Nouthern blotting
technique?
• …………………………………………
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Device
Numerical pH meter
Fast, Accurate, and
Auto Calibrated
instrument used for
adjustment of
pH

Letter Wlab methods
level 3
2017-2018

Tool
Wire gauze
It is a sheet of thin metal. wire
gauze sits on the iron ring to
provide a place to stand a
beaker.
on older wire gauze, the white
material was asbestos
currently it is a ceramic.

William Harvey
1578-1657
Researcher

William Harvey was a 17th-century. British physician who became
the first to document an understanding of blood circulation

William
Harvey
1578-1657
Harvey's research was furthered
through the dissection of
animals.
He published his theories in a
book entitled (Anatomical Study
of the Motion of the Heart and of
the Blood in Animals),
where he explained how the heart
propelled the blood in a circular
course through the body.

William
Harvey
1578-1657
Harvey was also the first to
suggest that humans and other
mammals reproduced via the
fertilization of an egg by
sperm.
It took a further two centuries
before a mammalian egg was
finally observed, but
nonetheless Harvey's theory
won credibility during his
lifetime

begins with a letter W?
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Technology or Technique or
Terms
“Western blotting”

Western blotting
(protein blotting or immunoblotting)
• Western blotting can produce qualitative and semi-
quantitative data about the protein of interest.
• It is an important technique used in cell and molecular
biology.
• It enables the researchers to identify the specific
protein from mixture of proteins extracted from cells as
well as evaluation of their size and amount.
• The SDS PAGE technique is prerequisite for western
blotting.

What’s mean the
SDS PAGE?
SDS-
PolyAcrylamide Gel
Electrophoresis

Western
blotting
steps
Tissue
preparation
Gel
electrophoresis
Transfer Blocking
Detection (two
step , one step)

Western blotting steps
•Proteins are extracted from the sample
•Proteins are separated by their sizes using
polyacrylamide gel electrophoresis (SDS-PAGE)
•Separated molecules are transferred into a *PVDF
membrane or nitrocellulose membrane by
electroporation
•The membrane is blocked for non specific binding with
the antibodies
*PolyVinylidene DiFluoride (PVDF) Membranes

Western blotting steps
•Transferred proteins are bound with primary antibody
(enzyme labeled antibodies).
•The membrane is washed to remove nonspecifically
bound primary antibodies
•Bound antibodies are detected by adding a substrate and
detecting the colored precipitate formed.

Western blotting Uses
➢ It is most sensitive and specific test for determining
size and amount of protein present in any material.
➢ The confirmatory HIV test employs a western blot to
detect anti-HIV antibody in a human serum sample.
➢ A western blot is also used as the definitive test for
Creutzfeldt-Jakob Disease, Lyme disease, Hepatitis B
infection and HSV-2 (Herpes Type 2) infection.

Advantages & Disadvantages
Advantages
Sensitivity
Because of its ability to detect as little as 0.1
ng of protein in a sample, the technique can
theoretically serve as an effective early
diagnostic tool, sensing even the slightest
immunogenic response from a virus or
bacteria in a patient sample.
Disadvantages
High cost
Technical Demand

previous study,
the western blotting
technique?
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Device
Water bath
A water bath is a device that
maintains water at a constant
temperature. It is used in the
Biotechnology laboratories

References
• http://www.bbc.co.uk/history/historic_figures/harvey_william.shtml
• https://www.slideshare.net/136659145/western-blot-
2563509?from_action=save
• http://www.microeguide.com/equipment/water_bath.asp
• https://www.slideshare.net/136659145/western-blot-
2563509?from_action=save

Letter D
Lab. methods ( )
Level 3
2017-2018

Tools
“Desiccator”
• Desiccators create and maintain dry
environments to ensure sample
stability.
• Using absorbing beads, and other
desiccants, desiccator cabinets or
apparatuses quickly absorb moisture,
guaranteeing sensitive materials do
not react to surroundings prior to
testing.

Researcher
(David Baltimore)
• Famous As: Virologist
• He is an American biologist who won a share of the 1975 Nobel Prize in
Physiology or Medicine. As a researcher, he has made tremendous
contributions to immunology, virology, cancer research, biotechnology, and
recombinant DNA research.
• he performed pioneering research on animal virology. Later in his career,
he independently discovered reverse transcriptase, an enzyme that
synthesizes DNA from RNA.
• And researched on interaction between viruses and the genetic
material of the cell.

begins with a letter D
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Technology or Technique
or Terms
“Differential display PCR”
“DDRT-PCR or DD-PCR”

Differential
display
definition
It is a laboratory technique that allows
a researcher to compare and identify
changes in gene expression at
the mRNA level between two or more
eukaryotic cell samples

Introduction
• Differential display is a rapid method which can be
applied to detect changes in the transcriptome in
response to environmental and temporal factors.
• Identification of mRNA, SnRNA and miRNA.

Principle of Differential display
Differential display relies on the use of two distinct
primers: an anchor primer and a second primer with an
arbitrarily chosen sequence. Anchor primers are used in both
the reverse transcription reactions and in the subsequent PCR
amplification while the second, arbitrary primer is used only
in the PCR reaction.

DD-RT PCR
technique
• Anchored primer
• Arbitrary primer

Troubleshooting Device:
1. Washing the two glass by 10% NaoH “leave it overnight for
removing all residual to No form bubbles” then double distal
H2O finally Ethanol absolute.
2. Machine composition: you should put binding solution on
glass “one of them”. Putting sigma coat on the other glass

Differential
display
• the rapidity and sensitivity of the assay.
• Does not require any prior knowledge of
the genome.
• Reproducibility.
• Speed and Expense.
Advantages
• Not easily automated or scaled-up.
• Non-specific and inefficient
amplification.
• Generate positive results unwanted.
Dis-advantages

Differential display applications
Identification of
transcripts that are
differentially
regulated.
1
Essential step
prior to carry out
whole
transcriptome
sequencing
2
Validation of RNA
transcripts isolated
from a whole
transcriptome
library
3

previous study,
the Differential display
technique?
• …………………………………………
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10

Device
Distilled
Water
Machine

Distilled Water Machine
• Equipment used for water purification and distillation includes
Deionized (DI) Water Systems, water distillers, reagent-grade
water systems, and laboratory filters. DI and distilled water are
the most common types of purified water used in the lab, but
techniques also used to produce high-purity water include: such
as; Carbon filtration.

Letter I
Lab. methods ( )
Level 3
2017-2018

Tools
“iris scissors”
• Iris scissors are a type of
scissors with short blades that
was originally developed for
ophthalmic surgery.
• Iris scissors are also available in
the crafting market and are
sometimes used for the
production of fabric-related
goods.

Researcher
(Isaac Asimov)
• Famous As: Writer, Professor
• Isaac Asimov is best known as the most successful writer of
science fiction and popular science books.
• Asimov opened the doors for the new age of science fiction
writing which the world had never tasted before him. Asimov is
credited with having edited over 500 books.

Researcher
(Isaac Asimov)
• Asimov was a brilliant professor of biochemistry at Boston
University. Besides being a prolific writer, Asimov was also an
integral part of (President) the American Humanist Association.
Asimov is also known for his work as a civilian at the
Philadelphia Navy Yard's Naval Air Experimental Station during
the World War II. “Robotics” was a term coined by Asimov
which went on to become a branch of technology.

Technology or
Technique or Terms
In Situ Hybridization
“ISH”

What’s mean in situ, in
molecular and cell biology?
In molecular and cell biology,
• in situ It means "locally", "on site", "on the
premises" or "in place" to describe an event
where it takes place, and is used in many
different contexts. For example, in fields such
as biology, in situ may describe the way a
measurement is taken, that is, in the same place
the phenomenon is occurring without isolating it
from other systems or altering the original
conditions of the test..

Introduction
•In situ hybridization (ISH) is a type of hybridization that uses a
labeled complementary DNA, RNA or modified nucleic acids
strand (i.e., probe) to localize a specific DNA or RNA sequence in a
portion or section of tissue (in situ), or, if the tissue is small enough
(e.g., plant seeds, Drosophila embryos), in the entire tissue (whole
mount ISH), in cells, and in circulating tumor cells (CTCs).
•It obtain temporal and spatial information about gene expression
and genetic loci

Scientific Basis of ISH
• Double-stranded DNA denatures on heating to single-stranded
DNA. On cooling, the single-stranded DNA reanneals with its
complementary sequence into double-stranded DNA.
• labeled fragment of a DNA sequence (a DNA probe) is
denatured and added to denatured nuclei or chromosomes on a
routine, air-dried interphase preparation during the process of
reannealing, some of the labeled DNA will hybridize to its
complementary sequence in the chromosomal DNA

ISH
principle
1. Denaturation
annealing
2. Labelling
examination

Types of In Situ Hybridization
• While the basic workflow of ISH is similar to that of blot
hybridizations. the nucleic acid probe is synthesized, labeled,
purified, and annealed with the specific target—the difference is
the greater amount of information gained by visualizing the
results within the tissue.
• There are two basic ways to visualize your RNA and DNA
targets in situ fluorescence (FISH) and chromogenic (CISH)
detection.

Types of In Situ Hybridization
Technique
Instrument/
visualization
method Primary advantage Primary application
CISH Bright-field
microscopy
Ability to view the CISH
signal and tissue
morphology
simultaneously
Molecular pathology
diagnostics
FISH DNA-
FISH
Fluorescence
microscopy
Multiplexable: visualize
multiple targets in the
same sample
Gene presence, copy
number, and location;
mutation analysis
RNA-
FISH
Fluorescence
microscopy, HCS, and
flow cytometry
Multiplexable: visualize
multiple targets in the
same sample
Gene expression, RNA
temporal and spatial
localization

1. Fluorescence In Situ
Hybridization
Multicolor FISH

2. Chromogenic In
Situ Hybridization (CISH)

Chromogenic In
Situ Hybridizati
on (CISH)
•Chromogenic in
situ hybridization (CISH)
is a cytogenetic technique
that combines the
chromogenic signal
detection method
of immunohistochemistry
(IHC) techniques with in
situ hybridization

Different between direct & indirect ISH
http://pubs.rsc.org/en/content/articlehtml/2014/ra/c3ra45401k

Directly labelled ISH probe
• Principle of in situ hybridization. This figure represents the
principles of ISH using either a directly- or indirectly- labelled
method. In directly-labelled ISH, the DNA probe is directly
conjugated to a fluorophore (yellow), as occurs in FISH.

Indirectly labelled
ISH probe
• In indirectly-labelled ISH, the DNA
probe is conjugated to a hapten
(pink). During the denaturation
step of the ISH protocol, the DNA
probe hybridizes to the gene of
interest. Hapten-conjugated probes
are detected either with a
chromogen-linked antibody (red)
(CISH), or with a fluorescently-
labelled antibody.

3. in situ
PCR
• in situ PCR is a
method in which the
polymerase chain
reaction actually
takes place in the cell
on a slide, and the
product can be
visualized in the same
way as in traditional
in situ hybridization.

3. in situ amplification
"In situ PCR”
• In situ PCR has mainly been used to identify DNA sequences
that are not easy to detect using standard in situ hybridization.
These sequences include human single‐copy genes,
chromosomal translocations, and rearranged cellular genes.
• In situ PCR is also used for mapping genomic sequences that
have a low copy number in metaphase chromosomes.
However, in situ PCR has multiple problems, including low
efficiency of amplification and poor reproducibility.

Catalyzed reporter deposition
(CARD)

4. Catalyzed reporter deposition
(CARD)
• This method involves deposition of activated biotinylated
tyramine onto electron rich moieties (e.g., tyrosine and
phenylalanine) at or close to the site of horseradish
peroxidase (HRP).
• When using HRP-labeled probes fluorescent staining results
from a secondary incubation with fluorescently labeled
tyramine.
• The specifically bound peroxidase molecules catalyze the
deposition of these labeled reporter compounds within cells
targeted by the HRP-tagged probe.

In Situ Hybridization steps:
https://greenfluorescentblog.wordpress.com/2013/02/21/rnascope-a-novel-fish-in-the-sea/

1. Preparation of Tissue
• Treatment with proteases (proteinase K is the most common)
• Acetylation of sections
• Optimization of tissue processing, including fixation and storage
2. In situ hybridization probes:
• Double-stranded DNA (dsDNA) probes
• Single-stranded DNA (ssDNA) probes
• RNA probes (riboprobes)

3. Labeling techniques:
• Radioactive probe
32P, 125I, 35S, or 3H
• Non-radioactive labels
• biotin
• digoxigenin
• fluorescent dye (FISH)

In Situ
Hybridization
example
• In situ hybridization
of wild
type Drosophila
embryos at different
developmental
stages for the RNA
from a gene called
hunchback.

InSitu
Hybridization
• Maximum use of tissue that is difficult to
obtain (e.g., embryos and clinical biopsies).
• Hundreds of different hybridizations can be
performed on the same tissue.
• Libraries of tissues can be formed and
stored in the freezer for future use.
Advantages
• Difficulty in identifying targets that have low
DNA and RNA copies. However, approaches
are continually being developed to improve
the sensitivity of in situ hybridization
Dis-advantages

Applications
of
In Situ
Hybridization
• Microbiology
• Pathology
• Developmental
biology
• Karyotyping and
phylogenetic
analysis
• Physical mapping

previous study,
the In Situ Hybridization
technique?
• …………………………………………
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10

Incubator Device
•In biology, an incubator is a device used to grow and
maintain microbiological cultures or cell cultures.
•The incubator maintains optimal temperature, humidity and
other conditions such as the carbon dioxide (CO2)
and oxygen content of the atmosphere inside.
•Incubators are essential for a lot of experimental work in cell
biology, microbiology and molecular biology and are used
to culture both bacterial as well as eukaryotic cells.

Letter M
Lab. Methods
Level 3
2017-2018

Calculation
SumitonPictureDescriptionName
Tool
Researcher
Technology
/Technique
Device
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DeviceTechnology
/Technique
ResearcherTool
-------/ 10-------/ 10-------/ 10-------/ 10Discussion

Tools
Magnetic Stir Bars
• A stir bar is
the magnetic bar
placed within the
liquid which
provides the stirring
action. The stir bar's
motion is driven by
another rotating
magnet or assembly
of electromagnets in
the stirrer device,
beneath the vessel
containing the liquid

Mention another Tools
begins with a letter M
• ………………………………………………………
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Researcher
Marguerite Vogt
Marguerite Vogt was a German-born, American cancer
biologist and virologist best known for her research on Polio
And Cancer at the ‘Salk Institute for Biological Studies’. She
collaborated with Nobel Prize-winning scientist Renato
Dulbecco to analyze the way polio virus develops plagues in
cell cultures, a discovery that eventually aided in the
development of a polio vaccine.
https://www.thefamouspeople.com/profiles/marguerite-vogt-7505.php

begins with a letter M
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Micro array
“cDNA microarray”
“Spotted Array”

Technology or Technique or Terms
“Micro array”
• The technique of microarrays came into use in the late 1990s.
Many cDNA clones were available from selected model
organisms.
• Many cDNAs can be spotted onto an array that is used to
determine changes in the level of expression of all cDNAs in
a comparison of two samples.

Introducing
DNA Microarray
•A DNA microarray consists of an orderly arrangement of DNA
fragments representing the genes of an organism. Each DNA
fragment representing a gene is assigned a specific location
on the array, usually a glass slide, and then microscopically
spotted (less than 1 mm) to that location. Through the use of
highly accurate robotic spotters, over 30,000 spots can be
placed on one slide.

Microarray uses
• DNA microarrays can be used to detect DNA (as
in comparative genomic hybridization), or detect RNA
(most commonly as cDNA after reverse transcription)
that may or may not be translated into proteins.
• The process of measuring gene expression via cDNA is
called expression analysis or expression profiling.

Affymetrix
“oligonucleotides chips”

Microarray chip
• A microarray is a glass microscope
slide containing thousands of
spots, either cDNA clones or
synthetic oligonucleotides.
• In the example below, each spot is a
single exon from a known gene.

The
Experiment
• comparison the
mRNAs of control
cells to mRNAs
from cells exposed
to
high temperature

Pipeline steps of Experiment:
RNA
extraction
cDNA
synthesis
Labeling
Hybridization
on Chip
Screening by
laser light
Data analysis

Aim of
assay
• The assay is actually
semiquantitative,
with a full range of
colors between red
and green showing
the relative
abundance of a
particular cDNA in
the two samples

Cluster
analysis
• In this example,
green is an increase
in expression in the
tumor relative to
control, while red is a
decrease. This is the
usual convention

Microarray
uses:
• Microarray analysis
can also be used to
identity new tumor
subtypes based on
patterns of gene
expression

Microarray
uses:
• Microarray analysis
can be used to
determine the
outcome of a
standard course of
treatment.

Microarray
uses:
• Differences in the
patterns of gene
expression in
tumors that appear
to be identical might
also be used to
identify drugs that
would be useful in a
particular case

Microarray applications
Gene
expression
profiling.
1
Chromatin
immunopreci
pitation
"CHIP"
2
SNP detection
3

Microarray
• Fast and easy to obtain results
• Different parts of DNA can be used to study gene expression.
• Huge step closer to discovering cures for diseases and cancer
Advantages
1.Correlations in results do not mean causation
2.It will the findings lead to unethical medical procedures
3.Very little knowledge is available about many genes
Dis-advantages

previous study,
• Mention, Different
method to
determination gene
expression?
• ……………………………
……………………………
……………………………
……………………………
…………………… 10

Device
Magnetic stirrer
• A magnetic stirrer or magnetic
mixer is a laboratory device that
employs a rotating magnetic
field to cause a stir bar (also
called "flea") immersed in a
liquid to spin very quickly, thus
stirring it.
• Magnetic stirrers are often used
in chemistry and biology

Letter S2
Lab. methods
Level 3
2017-2018

Tools
“Spatulas-lab”
• Laboratory double
spatulas in
stainless steel to
take a solid
(e.g. powder).
• Two flat spatulas
and a spoon
spatula.

Do you know different use for these?
• …………………………………
…………………………………
…………………………………
…………………………………
…………………………………
…………………………………
…………………………………
…………………………………
…………………………………
10

Researcher
(Salvador Edward Luria)
• Famous As: Microbiologist
• Who jointly won the Nobel Prize
in Physiology or Medicine in
1969 with Max Delbrück and
Alfred Hershey, for their
discoveries on the replication
mechanism and the genetic
structure of viruses.

Technology or
Technique or
Terms
“Sequencing”

Basic Methods of Sequencing
Maxam & Gilbert
Sequencing
“Chemically method”
Sanger Sequencing
Enzymatically
method

1. The Chemical
Sequencing
Method
Maxam & Gilbert

Maxam & Gilbert Sequencing
• This method based on chemical modification of DNA
and subsequent cleavage at specific bases.
• Maxam-Gilbert sequencing requires radioactive
labeling at one 5' end of the DNA and purification of the
DNA fragment to be sequenced.
• Chemical treatment then generates breaks at a small
proportion of one or two of the four nucleotide bases in
each of four reactions (G, A+G, C, C+T).

Maxam &
Gilbert
Sequencing
Preparation
Tube

Migration
• this method is based
on nucleobase-
specific partial
chemical modification
of DNA and
subsequent cleavage
of the DNA backbone
at sites adjacent to the
modified nucleotides

Reading Seq.

Movie
https://www.youtube.com/watch?v=lqWZ-duHfu8

Sanger
Sequencing
Method
“The chain-termination
Method”

Sanger Sequencing
• The target DNA is copied many times, making fragments of
different lengths. Fluorescent “chain terminator” nucleotides
mark the ends of the fragments and allow the sequence to be
determined.
• This separates the fragments by size, the smaller ones
traveling the furthest. Each of the four reactions are run in one
of four individual lanes, and the DNA bands are then visualized
by autoradiography or UV light. Then the DNA sequence can
be directly read on the X-ray film or gel, by reading up the
lanes.

Sanger Sequencing
Preparation Tube
• The classical chain-
terminator method
requires (DNA+
primer+ dNTPs) and
all modified di-
deoxynucleotidetrip
hosphates (ddATP,
ddGTP, ddCTP, and
ddTTP)

Sanger Sequencing
in vitro tube
• DNA fragments are
labelled with a
radioactive or
fluorescent tag on
the primer, in the
new DNA strand with
a labeled dNTP, or
with a labeled
ddNTP.

Sanger Sequencing
Reading Seq.

Sanger Sequencing
Reading Seq.
AutomaticallyManually

Maxam & Gilbert Vs. sanger seq.

Sanger
Sequencing
More recently, higher volume Sanger
sequencing has been supplanted
by "Next-Gen" sequencing methods,
especially for large-scale,
automated genome analyses.

Applications of sequencing
Molecular
biology
Metagenomics
Medicine
Forensics
Evolutionary
biology

1. Molecular biology
• Molecular biology: Sequencing is used to study genomes
and the proteins they encode. Information obtained using
sequencing allows researchers to identify changes in genes,
associations with diseases and phenotypes, and identify
potential drug targets.

2. Evolutionary biology
• Evolutionary biology: Since DNA
is an informative macromolecule in
terms of transmission from one
generation to another, DNA
sequencing is used
in evolutionary biology to study
how different organisms are
related and how they evolved.

• Metagenomics: The field of metagenomics involves
identification of organisms present in a body of water, sewage,
dirt, debris filtered from the air, or swab samples from
organisms.
3. Metagenomics

3. Metagenomics
• Metagenomics: Knowing which organisms are present in a
particular environment is critical to research
in ecology, epidemiology, microbiology, and other fields.
Sequencing enables researchers to determine which types of
microbes may be present in a microbiome, for example.

4. Medicine
• Medicine: Medical technicians may sequence genes (or,
theoretically, full genomes) from patients to determine if there
is risk of genetic diseases. This is a form of genetic testing,
though some genetic tests may not involve DNA sequencing.

previous study,
equipment needed to
conduct the experiment
transformation?
• …………………………………………
…………………………………………
…………………………………………
10

Device
scale
• balance designed to
measure
small mass in the sub-
milligram range.

Practical section of lab. method ppt 2017 2018

Practical section of lab. method ppt 2017 2018

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