Laboratory of Molecular Biochemistry
BIOC 432
2
Table of content:
Page Title Experiment no. 3 Rules of safety
4 Introduction to nucleic acids:
Structural properties.
Chemical and physical properties.
1
8 Extraction of DNA from blood by kit
(DNA purification)
2
11 UV &spectrophotometric analysis
of DNA &RNA
3
13 Estimation of DNA by diphenylamine 4
16 Extraction of RNA from blood
(RNA purification)
5
19 Extraction of DNA from strawberry 6
23 PCR & GEL ELECTROPHORESIS 7
31 Isolated of RNA from yeast
8
34 Estimation of RNA by orcinol
9
36
38
Estimation of total protein in serum (biuret reaction)
Isolation DNA from spleen
10
11
3
SAFETY RULES - Cell and Molecular Biology Laboratory
1. Laboratory excercises should be read before the laboratory period and work should be
planned.
2. Place bags, lab coats, books etc. in specified locations _NEVER ON THE BENCH TOPS
3. No eating or drinking in the laboratory. Do not store food in the laboratory.
4. No pipetting by mouth. Use mechanical pipetting devices only.
5. Wear laboratory coats, disposable gloves, and safety glasses when appropriate.
6. Use UV goggles and common sense when working with the UV lightbox.
7. Keep all noxious and volatile compounds in the fume hood.
8. Do not touch broken glassware with your hands. Use a broom and dustpan to clean it up.
Dispose off broken glass in appropriate receptacles. Do not toss out into regular trash.
9. Dispose of all biological waste into appropriate receptacles (Orange Biohazard bags).
Live cultures can be treated with Clorox bleach or autoclaved. Do not toss out into
regular trash or down drains without autoclaving.
10. Do not use plastic or polycarbonate containers, test tubes, pipettes etc. with phenol
and or chloroform. Instead use polypropylene or glass with these organic compounds.
Make sure to use gloves, goggles and lab coats when handling these chemicals.
11. Know the potential hazards of the materials, facilities, and equipment with which you
will work.
12. Know the location and proper use of fire extinguishers, eyewash stations, and safety
showers.
13. Do not dispose of hazardous or noxious chemicals in laboratory sinks. Use proper
containers in fume hood.
14. Report all accidents to the instructor immediately.
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Experiment1: Introduction to nucleic acids : structual properties
DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are polymers of nucleotides linked in
a chain through phosphodiester bonds. In biological systems, they serve as information-carrying
molecules.
Structure of nucleic acids:
Nucleotides are the building blocks of all nucleic acids. Nucleotides have a distinctive
structure composed of three components covalently bound together:
Nitrogen-containing "base" - either a pyrimidine {cytosine(C), thymine(T) and
uracil(U) or purine Adenine (A) and guanine (G)
Five-carbon sugar - ribose or deoxyribose
Phosphate group
Location and function of DNA:
Most DNA is located in the cell nucleus where it is called nuclear DNA, but a small amount
of DNA can also be found in the mitochondria where it is called mitochondrial DNA or
mtDNA. DNA serves as code for protein synthesis, cell replication and reproduction.
Function of RNA:
RNA normally occurs as a single-stranded molecule.
Essential function is to interpret DNA code and direct protein synthesis.
There are four types of RNA:
1) Transfer RNA (tRNA): carries amino acids in the cytoplasm to the ribosomes.
2) Messenger RNA (mRNA): re-writes DNA and takes it out of the nucleus to the ribosome.
3) Ribosomal RNA (rRNA): building blocks of ribosomes.
4) Small nuclear RNA (snRNA): refer to a number of small RNA molecules found in the
nucleus. These RNA molecules are important in number of processes including the
maintenance of the telomeres or chromosome ends.
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Chemical and physical properties of nucleic acids
Background:
DNA is generally stable than RNA, but there are many chemical and physical factors affect
the nucleic acids. The chemical factors that affect nucleic acids are such as hydrolysis by acids,
alkali, enzymes, and mutagenic factors of the DNA bases. The physical factors are heat, pH, salt
concentration, and base composition.
The ultraviolet absorption of nucleic acid:
Nucleic acids absorb in the ultraviolet region of the spectrum due to the conjugated double bond
and ring systems of the constituent purines and pyrimidines. The maximum absorbance is at the
wavelength 260 nm and minimum at 230 nm.
DNA hyperchromic & hypochromic effect:
The absorption of single strand DNA (ssDNA) is higher than the absorbance of double strand
DNA (dsDNA) this is known as a hyperchromic effect (means: “more color”). The hydrogen
bonds between the paired bases in the double helix limits the resonance behavior of the aromatic
ring of the bases which results in decrease in the UV absorbance of dsDNA (hypochromic
effect), while in ssDNA the bases are in free form and don't form hydrogen bonds with
complementary bases which results in 40% higher absorbance in ssDNA (hyperchromic) at the
same concentration.
The stability of DNA structure:
The stability of DNA structure depends on the integrity of two type bonds: phoshodiester
bonds (which link between the sugar and phosphate groups in the DNA backbone), this bond is
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Figure 1: DNA denaturation (breaking hydrogen
bonds) results in two separate strands. Figure 2: DNA digestion (breaking phoshodiester
bonds) results in DNA fragments.
very strong and can't be broken by conventional methods, it can be broken by specific nucleases
enzyme and hydrogen bond (which links between the complementary bases of the two
polynucleotide strands) are relatively weak and can be disrupted by different factors such as heat.
DNA denaturation & renaturation:
DNA denaturation, or DNA melting, is the process by which double-stranded DNA unwinds
and separates into single-stranded strands through the breaking of hydrogen bonds between the
bases. Complementary DNA reform is called annealing or renaturation. Disruption occurs in lab
by different methods such as: heating to high degree, change salt conc., adding alkali or change
pH.
dsDNA ssDNA
Strand separation,
denaturation, or
melting
Annealing,
renaturation, or
hyberdization
Helix Coil
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DNA denaturation by heating:
When DNA is heated, the temperature at which half of helix structure is lost is known as
melting temperature (Tm). The melting temperature depends on both the length of the DNA,
and the nucleotide sequence composition, higher GC content higher Tm. This is because the
triple hydrogen bonds between G and C need more energy to disrupt than the bouble bonds
between A and T.
Monitoring the DNA denaturation and recombination by UV absorbance:
When a solution of double-stranded DNA is slowly heated, the absorbance increases rapidly
to a higher value, which is not significantly changed by further heating. If the hot DNA solution
is then cooled slowly, the two threads recombine and the “cooling curve” should be
superimposed on the “melting curve”. If the DNA is cooled rapidly then some recombination of
the two strands takes place in a random manner so that the extinction of the solution at room
temperature is higher than the of the original DNA solution before heating.
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Experiment2: DNA Purification
PRINCIPLE:
Depending on the starting material, samples are digested with Proteinase K in either the
supplied Digestion or Lysis Solution. RNA is removed by treating the samples with RNase
A.
The lysate is then mixed with ethanol and loaded on the purification column where the DNA
binds to the silica membrane. Impurities are effectively removed by washing the column with
the prepared wash buffers. DNA is then eluted under low ionic strength conditions
with the Elution Buffer.
Step Procedure:
1- Add 400 μl of Lysis Solution and 20 μl of Proteinase K Solution to 200 μl of whole
blood, mix thoroughly by vortexing or pipetting to obtain a uniform suspension.
2- Incubate the sample at 56°C while vortexing occasionally or use a shaking water
bath, rocking platform or thermomixer until the cells are completely lysed (10 min).
3- Add 200 μl of ethanol (96-100%) and mix by pipetting or vortexing.
4- Transfer the prepared lysate to a DNA Purification Column inserted in a collection tube.
Centrifuge the column for 1 min at 6000 x g. Discard the collection tube containing the flow-
through solution. Place the DNA Purification Column into a new 2 ml collection tube (included).
5- Add 500 μl of Wash Buffer I (with ethanol added). Centrifuge for 1 min at 8000 x g.
Discard the flow-through and place the purification column back into the collection
tube.
6- Add 500 μl of Wash Buffer II (with ethanol added) to the DNA Purification Column.
Centrifuge for 3 min at maximum speed (≥12000 x g).
Optional. If residual solution is seen in the purification column, empty the collection
tube and re-spin the column for 1 min. at maximum speed.
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Discard the collection tube containing the flow-through solution and transfer the
DNA Purification Column to a sterile 1.5 ml microcentrifuge tube (not included).
7- Add 200 μl of Elution Buffer to the center of the DNA Purification Column membrane to
elute DNA. Incubate for 2 min at room temperature and centrifuge for 1 min at 8000 x g.
Note:
• For maximum DNA yield, repeat the elution step with additional 200 μl of Elution Buffer.
If more concentrated DNA is required or DNA is isolated from a small amount of starting
material (e.g., 50 μl) the volume of the Elution Buffer added to the column can be reduced to
50-100 μl. Please be aware that smaller volumes of Elution Buffer will result in smaller final
quantity of eluted DNA.
8- Discard the purification column. Use the purified DNA immediately in downstream
applications or store at -20°C.
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Result sheet
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Experiment3: UV Spectrophotometric Analysis of DNA and RNA
The concentration of an RNA or DNA sample can be checked by the use of UV
spectrophotometry. Both RNA and DNA absorb UV light very efficiently making it possible to
detect and quantify either at concentrations as low as 2.5 ng/µl. The nitrogenous bases in
nucleotides have an absorption maximum at about 260 nm. Using a 1-cm light path, the
extinction coefficient for nucleotides at this wavelength is 20. Based on this extinction
coefficient, the absorbance at 260 nm in a 1-cm quartz cuvette of a 50µg/ml solution of double
stranded DNA or a 40µg/ml solution of single stranded RNA is equal to 1. You can calculate
the concentration of the DNA or RNA in your sample as follows:
DNA concentration (µg/ml) = (OD 260) x (dilution factor) x (50 µg DNA/ml)/(1 OD260 unit)
RNA concentration (µg/ml) = (OD 260) x (dilution factor) x (40 µg RNA/ml)/(1 OD260 unit)
In contrast to nucleic acids, proteins have a UV absorption maximum of 280 nm, due mostly to
the tryptophan residues. The absorbance of a DNA sample at 280 nm gives an estimate of the
protein contamination of the sample. The ratio of the absorbance at 260 nm/ absorbance at 280
nm is a measure of the purity of a DNA sample; it should be between 1.65 and 1.85.
Phenol has an absorbance maximum of 270 but the absorbance spectrum overlaps considerably
with that of nucleic acids. If there is phenol contamination in your DNA sample, the absorbance
at 260 nm will be high, giving a false measure of DNA concentration. These procedures are
specific to the Beckman DU 640B spectrophotometer.
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Result sheet
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Experiment4: Estimation of DNA by diphenylamine
Principle:
The DNA is treated with diphenylamine under acidic condition; a blue compound is formed
with a sharp absorption maxima at 595 nm. This reaction is given by 2-deoxypentoses in general
and not specific for DNA. In acid solution, the straight chain form of the deoxypentose is
converted to highly reactive hydroxy levuinaldehyde which reacts with diphenylamine (D.P.A.)
to give a blue complex. In DNA only the deoxyribose of the purine nucleotides reacts so that the
value obtained represents half of the total deoxyribose present.
Materials:
Calf thymus DNA standard 1000 ug/ml (2 ml) for every student.
D.P.A. reagent (1g of pure diphenylamine + 100 ml glacial acetic acid + 2.5 ml conc.
sulphuric acid). All the reagents are made fresh. Note: D.P.A is a poisoning reagent.
Equipments:
Spectrophotometer
Pipettes (1 ml & 5 ml).
5 x 10 ml test tubes.
Water bath
Parafilm
Procedure:
1. Pipettes (0.2, 0.3, 0.4 and 0.5 ml) of standard DNA & 0.3 ml of unknown into test tubes,
complete to 1.5 ml with distill water (D.W) and mix.
2. Add 2.5 ml of D.P.A., and then mix the mixture.
3. Place tubes in 100°C water bath for 10 min.
4. Cool the tubes and measure the Optical density (O.D.) at 595 nm.
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Follow the below procedure for doing the experiment.
Reagents 1 2 3 4 5
Standard DNA
(1000 μg/ml) 0.2 ml 0.3 ml 0.4 ml 0.5 ml ---
Unknown --- --- --- --- 0.3 ml
H2O 1.3 ml 1.2 ml 1.1 ml 1.0 ml 1.2 ml
D.P.A 2.5 ml 2.5 ml 2.5 ml 2.5 ml 2.5 ml
Total 4 ml 4 ml 4 ml 4 ml 4 ml
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Result sheet
A. Calculate the conc. of each standard DNA and record your results in table:
Tube 1 2 3 4 5 unknown
Conc.
(μg/ml)
Abs.
B. Drawing a standard curve using the absorbance readings against the conc. (μg/ml), then
determine the conc. unknown of DNA, then the result multiple by 2.
C. Calculate conc. unknown (μg/ml & μg%).
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Experiment5: RNA Purification
PRINCIPLE:
Samples are lysed and homogenized in Lysis Buffer, which contains guanidine thiocyanate, a
chaotropic salt capable of protecting RNA from endogeneous RNases (1). The lysate is then
mixed with ethanol and loaded on a purification column. The chaotropic salt and ethanol cause
RNA to bind to the silica membrane while the lysate is spun through the column (2).
Subsequently, impurities are effectively removed from the membrane by washing the column
with wash buffers. Pure RNA is then eluted under low ionic strength conditions with
nucleasefree water.
Procedures:
1. Collect blood cells by centrifugation of 0.5 ml of whole blood at 400 x g for 5 min at 4ºC.
Blood cells will generate a pellet of approximately 6070% of the total sample volume.
Remove the clear supernatant (plasma) from the pellet with a pipette.
2- Resuspend the pellet in 600 μl of Lysis Buffer supplemented with (β-mercaptoethanol or
DTT). Vortex or pipet to mix thoroughly.
3- Add 450 μl of ethanol (96100%) and mix by pipetting.
4- Transfer up to 700 μl of lysate to the RNA Purification Column inserted in a collection tube.
Centrifuge the column for 1 min at ≥12000 x g. Discard the flow-through and place the
purification column back into the collection tube. Repeat this step until all of the lysate has been
transferred into the column and centrifuged. Discard the collection tube containing the flow
through solution. Place the RNA Purification Column into a new 2 ml collection tube (included).
5- Add 700 μl of Wash Buffer 1 (supplemented with ethanol) to the RNA Purification Column
and centrifuge for 1 min at ≥12000 x g. Discard the flow-through and place the purification
column back into the collection tube.
6- Add 600 μl of Wash Buffer 2 (supplemented with ethanol) to the RNA Purification Column
and centrifuge for 1 min at ≥12000 x g.Discard the flowthrough and place the purification
column back into the collection tube.
7- Add 250 μl of Wash Buffer 2 to the RNA Purification Column and centrifuge for 2 min at
≥12000 x g. Optional. If residual solution is seen in the purification column, empty the collection
tube and respin the column for 1 min. at maximum speed.Discard the collection tube containing
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the flow-through solution and transfer the RNA Purification Column to a sterile 1.5 ml
RNasefree microcentrifuge tube (included).
8- Add 50 μl of Water, nucleasefree (included) to the center of the RNA Purification Column
membrane. Centrifuge for 1 min at ≥12000 x g to elute RNA.
9- Discard the purification column. Use the purified RNA for downstream applications or store
RNA at 20°C or 70°C until use.
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Result sheet
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Experiment6: DNA Extraction from strawberries
Background Knowledge:
The soap is to dissolve the lipid bilayer around the cell and
nucleus. The salt is to neutralize the negative charge of the DNA. The alcohol is used
because DNA is soluble in water but not soluble in alcohol. The bubbles on the DNA in
the alcohol layer are just dissolved gasses and are not part of the DNA.
Summary:
Students will extract and compare DNA from both bananas and strawberries.
Goals & Objectives: Students will be able to experience how DNA looks the same from
one organism to another. Students will be able to describe how genetic engineering is
important in today’s society.
Standards: CA Biology 5a. Students know the general structures and functions of DNA,
RNA, and protein. 5c. Students know how genetic engineering (biotechnology) is used to
produce novel biomedical and agricultural products.
Time Length: 60 minutes
Prerequisite Knowledge: Students have been introduced to cell organelles and know
that DNA has the same structure in all organisms.
Pre-Lab
Set-up stations: alcohol station with ice-cold alcohol and a buffer station with two
graduated cylinders. The buffer should be made using a large flask and then be poured
into a 100mL beaker.
Accommodations: Students with an IEP can take the handout home if they need extra
time but must finish the lab procedures in class.
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Materials:
• Strawberries (fresh or thawed), and fresh bananas • Cheesecloth
• Small funnel • 90% Ethanol ice-cold
• Graduated cylinders • Large test tubes
• Zip-lock freezer bags • 1L Erlenmeyer flask and 100 mL beaker
• 10 mL graduated cylinder
• 7 mL DNA buffer (50 mL dish soap-15 g salt-900 mL tap water)
• Glass stirring rod
• Safety goggles
Tris buffer pH8:(5ml of 1M Tris-Hcl,pH7.6+2ml of 0.5M EDTA,pH8/1Lwith H2O,pH8)
EDTA(0.5M):0.18g/1ml
TRIS(1M):1.21g/10ml
Procedures:
1. In groups of 3: one student is the assistant (gets buffer solution, hold funnel while
pouring juice into a test tube, and put away materials), one student is in charge of
extracting the strawberry DNA, and the last student is in charge of extracting the
banana DNA.
2. Place one strawberry in a zip-lock bag, press the air out, then seal it. Softly mash
the strawberry/banana with your fingers until it becomes a juice puree (1-2 minutes).
3. Add 8 mL of buffer to the strawberry/banana and then press the air out of the bag
and seal.
4. Mash the strawberry/banana carefully for 1 minute without creating many
bubbles.
5. Place the test-tube in a cup. Put the funnel on top of the test-tube. Place the
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cheesecloth on top of the funnel.
6. Open the bag and drain carefully the strawberries/bananas on top of the
cheesecloth to fill the test-tube with ¼ juice. The juice will drain through the
cheesecloth but the chucks of strawberries/bananas will not pass through into the
test-tube.
7. Tilt the test-tube and pour in an equal amount of alcohol, ¼ of test-tube, through
the funnel and down the sides of the test-tube. This will allow for better
separation of the DNA.
8. Place the test-tube so that it is eye level. Using the stirring rod, collect DNA at the
boundary of alcohol and strawberry juice. Do not stir the strawberry/banana juice;
only stir in the above alcohol layer.
9. Gently remove the stirring rod and examine what the DNA looks like. Clean up
using the teacher’s instructions after you have finished the lab write-up.
10.Then, collect the DNA in eppendorf tube and add 0.5ml Tris buffer(mix).
11.store at -20 ºC.
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Result sheet
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Experiment7: PCR & GEL ELECTROPHORESIS
DNA structure:
Nucleus is the “command center”
Chromosomes
Double-stranded
DNA helix
“genetic code”
Design of work:
DNA extraction PCR (amplification of a gene) RFLP-restriction digestion of a
gene visualization of result in gel electrophoresis
PCR –Amplification of nucleic acid:
Principle:
The purpose of a PCR ( polymerase chain reaction ) is to make a huge number of copies of a gene
that without PCR it would be undetectable.
Polymerase:
-DNA polymerase duplicates DNA.
Chain Reaction:
-The product of a reaction is used to amplify
the same reaction.
-Results in rapid increase in the product.
Properties of DNA polymerase:
Needs a pre-existing DNA to duplicate
Called template DNA
Can only extend an existing piece of DNA
Called primers
DNA polymerase needs Mg++ as cofactor
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Each DNA polymerase works best under optimal temperature, pH and salt concentration
PCR buffer provides optimal pH and salt condition
DNA strands are anti-parallel
One strand goes in 5’ 3’
The complementary strand is opposite.
DNA polymerase always moves in one direction (from 5’ 3’).
DNA polymerase incorporates the four nucleotides (A, T, G, C) to the growing chain.
dNTP follow standard base pairing rule.
The newly generated DNA strands serve
as template DNA for the next cycle
PCR is very sensitive
Widely used
PCR reaction components:
Water
Buffer
DNA template
Primers
Nucleotides (dNTPs)
Mg++ ions
DNA polymerase (Taq polymerase)
Taq DNA polymerase:
- Derived from Thermus aquaticus
- Heat stable DNA polymerase
- Ideal temperature 72ᵒC
Thermal Cycling: PCR machine controls temperature
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Copies of DNA=2N
Restriction digestion:
DNA is prepared by digestion with restriction enzymes.
The sections of DNA that are cut out are called restriction fragments.
This yields thousands of restriction fragments of all different sizes.
Gel Electrophoresis
Electro = flow of electricity phoresis (from the Greek) = to carry across
Is a technique that is used to separate charge molecules especially proteins and nucleic acids as DNA,
RNA that differ in size, charge or conformation.
{A gel is a colloid, a suspension of tiny particles in a medium, occurring in a solid form, like gelatin}
A method used in biochemistry and molecular
biology to separate DNA or RNA
Principle:
• Under the influence of electrical field, charged molecules will migrate toward the electrode
that carry an opposite charge.
• Nucleic acids that have negatively charged will moving through a gel matrix toward the
anode (positively charge).
• Shorter molecules move faster and migrate farther than longer ones.
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.
Requirement of gel electrophoresis:
• Gel "supporting media".
• Buffer.
• Fluorescent dye.
• Samples
• DNA Marker.
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• Electrophoresis apparatus: Tank, plate, electrodes, power supply, and combs.
• Detection system.
Gel:
There are two types of gel:
Agarose :
• It is a polysaccharide extracted from seaweed .
• Polymerized agarose is porous, allowing the movement of DNA through it.
• It can be separate from about 0.2 kbp to 50 kbp of DNA fragments.
• Agarose gels have a large range of separation, but relatively low resolving power.
polyacrylamide.
• It is a cross-linked polymer of acrylamide.
• A wide range of conc. can be used between 3.5% to 20% (Advantage to give higher
resolution to separate very small DNA fragments that are differ in a one bp and so used for
DNA sequencing).
• Polyacrylamide gels are more annoying to prepare than agarose gels and neurotoxin
(Disadvantage).
• Polyacrylamide gels have a small range of separation, but very high resolving power.
Buffers:
Two buffers are used together:
Electrophoresis buffer:
• Provide ions to conduct the electricity and to maintain the pH at constant value.
• TBE buffer (Tris/Borate/Na2EDTA) is usually used.
Loading buffer:
• Others names: Tracking buffer and blue juice.
• It is used a color marker and density to the sample when load into wells.
• For example, bromophenol blue.
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Fluorescent dye:
• It is important to visualize the separated DNA bands
• usually ethidium bromide (EtBr) is used.
• EtBr is a fluorescent that intercalating between the bases of DNA and glows pink when
excited by UV dye.
Samples:
• It can be DNA, RNA and Protein.
DNA Molecular weight Marker:
• Others names: DNA Molecular weight Marker, DNA ladder, or DNA standard.
• It is a mixture of DNA fragments of known sizes.
• The size of a fragment is measured by base pairs (bp).
Electrophoresis apparatus:
• Tank: It is the container which contain the buffer.It always has a cover to prevent the
• Tray: Is the actual mold which provides a shape for the gel as it polymerizes (or solidify).
evaporation of buffer and for safety.
• Power supply
• Combs: It used to make wells on the gel to load different samples.
Detection system:
• Transilluminator (Ultraviolet light box) : to visualize the bands.
Pic:
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Application
� Estimation of the size of DNA molecules
� Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
Factors affecting migration
1) DNA or RNA molecular weight.
2) Voltage.
3) Agarose.
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Result sheet
31
Experiment8: Isolated of RNA from yeast
Principle:
Yeast is eukaryotic microorganisms that belong to the kingdom of fungi; there are 100,000
species or more. This experiment Saccharomyces cerevisiae (Baker’s yeast) is used in baking. It
is one of the most studied eukaryotic model. It reproduces by division process known as
budding. Many proteins in human biology were first discovered by studying their homologs in
yeast. S. cerevisiae was the first eukaryotic genome to be completely sequenced. The genome
composed of 13,000,000 base pairs, 6275 genes only 5,800 genes are functional. It was estimated
that yeast shares 23% of its genome with humans.
Total yeast RNA is obtained by extracting a whole cell homogenate with phenol. The
concentrated solution of phenol disrupts hydrogen bonding in the macromolecules, causing
denaturation of the protein. The turbid suspension is centrifuged and two phases appear: the
lower phenol phase contains DNA, and the upper aqueous phase contains carbohydrate and
RNA. Denatured protein, which is present in both phases, is removed by centrifugation. The
RNA is then precipitated with alcohol. The product obtained is free of DNA but usually
contaminated with polysaccharide. Further purification can be made by treating the preparation
with amylase.
Materials:
anol
Equipment:
Procedure:
1. Suspend 2.5 g of dried yeast in 15 ml of water previously heated to 37°C. Leave for 15 min at
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this temperature and add 12.5 ml of concentrated phenol solution (Care: corrosive).
2. Stir the suspension mechanically for 30 min at room temperature, then centrifuge at 5000 rpm
for 15 min in the cold to break the emulsion.
3. Carefully remove the upper aqueous layer with a Pasteur pipette and centrifuge at 5000 rpm
for 7 min in a refrigerated centrifuge to sediment denatured protein.
4. Add potassium acetate to the supernatant to a final concentration of 20 g/litre (Note: every 1
ml of supernatant adds 9 ml of potassium acetate) and precipitate the RNA by adding 2
volumes of ethanol.
5. Cool the solution in ice and leave to stand for 1 h.
6. Collect the precipitate by centrifuging at 5000 rpm for 7 min in the cold.
7. Wash the RNA with ethanol-water (3:1) depend on the amount of precipitate.
8. Filter the solution and then add 3 ml of ethanol to the filter paper.
9. Finally, wash with 3 ml ether; air dry, and weight. (Note: Yeast contains about 4 per cent
RNA by dry weight).
10. Dissolve RNA powder in 10 ml, 1% NaOH.
11. Compare your product with a commercial preparation by measuring the pentose, phosphorus,
and DNA content and determining the absorption spectrum. Keep your preparation for use in
later experiments.
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Result sheet
Hint: Yeast contains about 4 percent RNA by dry weight
1. Calculate the weight of RNA?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
2. What is the yield of RNA?
______________________________________________________________________________
______________________________________________________________________________
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Expemint 9. Estimation of RNA by orcinol
Principle:
This is a general reaction for pentose. The orcinol reagent reacts with pentose
group in the backbone of the RNA molecules and depends on the formation of
furfural, when the pentose is heated with concentrated HCL. Orcinol reacts with
the furfural in the presence of ferric chloride act as a catalyst to give a green color.
Only the purine nucleotides give any significant reaction.
Materials:
Orcinol reagent (6% orcinol reagent in 100 ml ethanol)
Ferric chloride + HCL solution (0.5 ml of 10% ferric chloride solution was
added to 99.5 ml of conc. HCL)
Sat. RNA 250 μl/ml
All reagents are made fresh
Method:
Add the following amount in 3 tubes.
St. Unk. blank
St. RNA solution 1ml -- --
Unk. RNA solution -- 1ml --
D.W -- -- 1ml
orcinol 0.2 ml 0.2 ml 0.2 ml
Fecl3+HCL 3 ml 3 ml 3 ml
Place the tubes in 100 C˚ for 30 min.
Cool and read at 660 nm.
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Calculation:
C unk. = A unk. . C st.
A st.
Result sheet
36
Experiment10: Estimation of total protein in serum (biuret reaction)
Principle:
Substances which contain two CONH2 group joined together directly and those which contain
two or more peptide links. Give a blue to purple colored compound with alkaline copper
solutions.
It is composed of CuSo4; gives Cu++ ion in soln.
KI; prevent the oxidation of cuprous biuret.
Procedure:
components T (ml) St (ml) B (ml)
Biuret reagent 5 5 5
Serum or plasma 0.1 - -
St. BSA (5g/dl) - 0.1 -
D.W - - 0.1
Mix and incubate at 37°C for 10 min or at room at room temp for 20 min.
Read test and St against blank at 546 nm.
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Calculation:
Total protein conc. (g/dl) = ( ODT / ODSt )*St. Conc.
Result sheet
38
Experiment11: Isolation DNA from spleen
Principle:
Although almost all cells contain DNA, the amount present in some tissues is quite small so that
they are not a particularly convenient source. In addition, some tissues contain high
deoxyribonuclease activity so that the DNA is broken down into smaller fragments. A
convenient source for the isolation of DNA should therefore contain a high quantity of the
material and have low deoxyribonuclease activity.
Lymphoid tissue is very good in these respects and thymus is probably the best source, with
spleen as a good alternative. The tissue is homogenized in isotonic saline buffered with sodium
citrate pH 7.4. At this ionic strength, the deoxyribonucleoprotein is insoluble and separates well
from other proteins. Sodium citrate inhibits deoxyribonuclease activity by binding Ca and
Mg ++ , which are cofactors for this enzyme. The extraction procedure is carried out in the cold
so that any residual DNA'ase activity is minimal. Glass or plastic vessels are used throughout to
avoid degradation of the DNA.
The DNA is finally precipitated as a fibrous white mass by the addition of ethanol. After
washing with ethanol, the DNA is dissolved in saline buffered with sodium citrate to pH 7.4.
The material is best stored frozen and does not undergo any demonstrable change for several
months but drying of the DNA tends to lead to denaturation.
Materials:
Method:
1. Chop 5 g of calf spleen into small fragments and homogenize with 20 ml of buffered saline
for 1 min.
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2. Centrifuge the suspension at 5000 g for 15 min
3. Rehomogenize the precipitate in a further 40 ml of buffered saline.
4. Discard the supernatant and suspend the combined sediments uniformly in 2 mol/litre NaCl
to a final volume of 100 ml when most of the material should dissolve.
5. Remove any sediment by centrifugation and stir the solution continuously with a stirring
glass rod while adding an equal volume of ice-cold water.
6. Spool the fibrous precipitate on to a glass rod and leave it to stand in a beaker for 30 min.
During this time the clot will shrink and the liquid expressed should be removed with filter
paper.
7. Dissolve the deoxyribonucleoprotein in about 100 ml of 2 mol/litre NaCl.
8. Add an equal volume of the chloroform/amyl alcohol mixture (6:1), and blend for 30s.
9. Centrifuge the emulsion at 5000 g for 10-15 min and collect the upper (opalescent) aqueous
layer containing the DNA. This is best carried out by gentle suction into a suitable container
so that the denatured protein at the interface of the two liquids is not disturbed.
10. Repeat the treatment with organic solvent twice more and collect the supernatant in a 500 ml
beaker.
11. Precipitate the DNA by slowly stirring 2 volumes of ice-cold ethanol with the supernatant
and collect the mass of fibres on the glass stirring rod.
12. Carefully remove the rod and gently press the fibrous DNA against the side of the beaker to
expel the solvent.
13. Finally, wash the precipitate by dipping the rod into a series of solvents and expelling the
solvent as described. Four solvents are used: 70 per cent v/v ethanol, 80 per cent v/v ethanol,
absolute ethanol and ether. Remove the last traces of ether by standing the DNA in a fume
cupboard for about 10 min.
14. Weigh the dry DNA and dissolve by continuously stirring in buffered saline diluted one in
ten with distilled water (2 mg/ml); store frozen until required.
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