Laboratory of Molecular Biochemistry

BIOC 432

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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

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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

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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

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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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