COLLEGE OF NATURAL AND COMPUTIONAL SCIENCE

DEPARTMENT OF BIOLOGY

MSc applied Microbiology

COURSE TITLE: Molecular Techniques in Biology

Course code: 548

LABORATORY REPORT: In Animal blood cell of DNA isolation protocol

Name: Melese Endrias ID: 0006/13

TITLE: ANIMAL BLOOD CELL DNA ISOLATION PROTOCOL

OBJECTIVES

-TO KNOW ANIMAL BLOOD CELL DNA ISOLATION PROTOCOL.

SUBMITTED TO: KEDIR WOLIY (PhD)

June, 2022,

Hawassa, Ethiopia
Introduction

A laboratory is a facility that provides controlled conditions in which scientific research,

Experiments and measurement may be performed. The name, type and facility of the
laboratory differ based on the specific requirements of a scientific field. A technique in
molecular biology laboratory is a facility where techniques in molecular biology are used to
study the molecular basics of life and manipulate as desired. We have visited animal
biotechnology laboratory of Hawassa College of Agriculture and we have done some
experiments works( such as Isolation of DNA from Animal cell and tissue, Quantitative Analysis
of DNA, PCR Amplification and Qualitative Analysis of DNA). A general method for the isolation
of total DNA from animal tissue is described below. It is modified from the original procedure
described by Gross-Bellard et al using proteinase K, SDS and phenol! Chloroform. First, tissue or
the whole body of a small organism is rapidly frozen in liquid nitrogen and ground to a fine
powder. The processed tissue is then suspended in an extraction buffer containing proteinase
K, 50s and EDTA which are used to digest cellular proteins or inactivate nucleases. By successive
phenol Chloroform extractions, the mixture containing DNA is deproteinized and the DNA
finally recovered by ethanol precipitation. Here, and also in the other methods described later,
ammonium acetate is used in DNA precipitation instead of sodium acetate, because the
precipitation can occur at room temperature and DNA precipitated by ammonium salt seems to
be more digestible. This procedure has been chosen (from among many other alternatives)
because it is a well-recognize procedure for isolating pure and high molecular weight DNA. DNA
obtained in this way is generally free from protein and nucleases and is stable when stored at
4°C. Before starting DNA extraction, it is recommended that the 'Notes' are carefully read.
Cross-contamination is a big concern during parallel extractions, especially for population
biology studies employing PCR-related techniques. It is therefore important that blank
extraction controls be carried out along with normal DNA extractions to check for
contamination, and that all glassware, plastic ware and buffers/solutions used in DNA isolation
are autoclaved or sterilized. Hydrochloric acid treatment may be used to break down foreign
DNA on mortars, pestles and other glassware. The bench surface where extraction is carried out
should be cleaned with detergent, water and ethanol and should not be used for subsequent
PCR work. A set of clean instruments (mortar, pestle, scalpel, pipette, Pasteur pipettes, gloves,
etc.) should be used for each sample (each extraction) and gloves should be changed when
handling a different sample.

Experiment No.1

Objective: Isolation of DNA from Animal Blood cell and tissue this laboratory works were
aimed to:

 Acquaint a set up and safety of the techniques in molecular biology laboratory.

 Familiarize different techniques in molecular biology laboratory equipment’s and their
utility.

Materials and Chemicals: At That Time our Experiments works we have used the following
materials and chemicals:

Materials:
 Vortex
 PCR
 Glove
 Computer  Spatulas
 Comb  Balance
 Agarose gel chamber
 Centrifuge  Centrifuge
 Micropipette  Silica gel

 Microcenterfuge

Chemicals:
o Sheep blood cell o DNA Wash Buffer
o BL Buffer o Phenol
o Ethidium Bromide
o HBC Buffer o Water
o Elution Buffer o Loading buffer
o Sodium acetate
o 1oo%Ethanol
o Agarose powder
Procedure:

1 The sample was transferred into a sterile micro centrifuge tube and bring the volume up
to 250µl with Elution buffer
2 25µl OB Protease solution and 250µl with BL Buffer
3 Preparation was incubated at 65◦c for 10 minutes
4 260µl of 100% ethanol was added
5 Centrifuged briefly
6 Inserted in to a 2ml collection
7 The enter sample was transferred to column
8 Centrifuged at ≥10,000xg for 1 minute
9 The filtrate the collection tube were discarded
10 DNA mini column was inserted in to a new 2ml collection tube
11 500µl HBL Buffer diluted with 100% isopropanol was added, centrifuged at ≥10,000xg
for 1 minute. Filtrated was discarded and collection tube reused
12 700µl DNA wash buffer diluted with ethanol was added, centrifuged at ≥10,000xg for 1
minute. Filtrated was discarded and collection tube added.
13 The sample was repeated step 12 for a second DNA wash buffer wash step.
14 HiBind DNA mini column at maximum speeded for 2 minutes to dry the column.
15 HiBind DNA mini column was transferred into a nuclease-free 2ml microcenterifuge
tube
16 100-200µl Elution buffer was heated to 65⁰c
17 Step 16 was repeated for a second elution step
18 Eluted DNA was stored at -20⁰c

Experiment 2 Quantitative Analysis of DNA

Objective: to separate and visualize DNA bands by Agarose gel electrophorese

Introduction:

Agarose gel electrophoresis is a powerful and widely used method that separates molecules on
the basis of electrical charge, size, and shape. The method is particularly useful in separating
charged biologically important molecules such as DNA (deoxyribonucleic acids), RNA
(ribonucleic acids), and proteins. Agarose forms a gel like consistency when boiled and cooled
in a suitable buffer. The agarose gel contains molecule sized pores, acting like molecular sieves.

The pores in the gel control the speed that molecules can move. DNA molecules possess a
negative charge in their backbone structure due to the presence of PO4- groups thus this
principle is exploited for its separation. Smaller molecules move through the pores more easily
than larger ones. Conditions of charge, size, and shape interact with one another depending on
the structure and composition of the molecules, buffer conditions, gel thickness, and voltage.
Agarose gels are made with between 0.7% (provides good resolution of large 5–10 kb DNA
fragments) and 2% (good resolution for small 0.2–1 kb fragments). The gel setup provides wells
for loading DNA in to it. The loaded DNA molecules move towards the positively charged
electrode (anode) and get separated along the length of the gel. Ethidium bromide (EtBr), a
chromogen is added to the gel to visualize the separated DNA under UV transillumination. EtBr
intercalates between the bases and glows when UV radiation is passed through the gel.

Factor affecting migration of DNA in agarose gel:

• Size of the DNA

• Agarose concentration

• Conformation of DNA

• Voltage applied

• Composition of electrophoresis buffer

• Presence of intercalating dye

• Direction of electric field

• Base composition and temperature

PROCEDURE:

1 The Measured 1gm of Agarose powder buffer

2 100ml of TBE was added to Agarose powder buffer
3 Mixed and boiled by hot plate stirrer
4 EtBr 0.5µl was added after cooled
5 Poured on to gel tray and stayed until solidified
6 Running buffer was added
7 Sample was loaded added on the well carefully
8 The band were visualized

RESULTS AND OBSERVATION

From the experiment we have gotten the following results:

 DNA molecule was extracted. It was a colorless liquid in a tube.

 We have seen clear, separate bands of DNA fragments in a visualizing computer

During the lab work we have observed and learnt about the materials utilized in Animal
Biotechnology laboratory of Hawassa University, college of agriculture.

The materials were: Equipments in molecular biology laboratory

Magnetic Stirrer & Vortex

Magnetic stirrer: is a device which provides mixing and keeping the chemical solutions and

mixtures at a certain time and temperature by the help of a magnetic bar.

Vortex: agitates the solutions in the tube, flask and so on in certain speed and duration.

Agarose gel electrophoresis

Agarose gel electrophoresis is a powerful and widely used method that separates molecules on
the basis of electrical charge, size, and shape. The method is particularly useful in separating
charged biologically important molecules such as DNA (deoxyribonucleic acids), RNA
(ribonucleic acids), and proteins. Agarose forms a gel like consistency when boiled and cooled
in a suitable buffer. The agarose gel contains molecule sized pores, acting like molecular sieves.
Gel Documentation System (UVP)

This device is used to display DNA fragments after electrophoretic run. This instrument can also

be associated with the camera, and computer facilities to capture gel image for publication; in
such case it is termed as a gel doc system.

Spectrophotometry machine: A photometer for measure the relative intensities of the light in
different parts of a spectrum.
PCR (polymerase chain reaction)

This device is used for the amplification of a specific region of any DNA sample with

polymerase chain reaction in a test tube. It is also used for detection and constitution of

genetically modified organisms, as well as other genetic analyses. This device is used for the
amplification of a specific region of any DNA sample with polymerase chain reaction in a test
tube. It is also used for detection and constitution of genetically modified organisms as well as
other genetic analyses.
Thermo cycler (PCR machine)
Xylene cyanol gives a greenish blue color while bromophenol blue provides bluish colored zone.

The successful DNA run is determined by the presence of both the colored dye in the gel.

Hhm/
Discussion

Obtaining intact DNA is key to accurate sequence results. Animal cell tissues are best source for higher
molecular weight DNA. freezing with dry ice, or cooling ice helps to maintain integrity of tissue and the
DNA .in many cases rapid desiccation with slice gel has made it possible to transport sample back to
laboratory for successful DNA extraction and analysis. However, for some species and tissues this
method has been less than ideal leading to acceptable low yielding and/or degraded DNA.

Conclusion

-In generally the method and equipment’s tested yield high quality DNA under laboratory condition. The
laboratory nucleic acid extraction method should prove useful for working in remote sites, there ice ,dry
ice are unavailable when degradation is likely to occur due long distance between the sample site and
the laboratory ,when high molecular weight DNA is needed and for cases where other
collection ,preservation and extraction methods have been effective.

-The 260/280ratio was used to determine protein contamination of nucleic acid sample.

-260/230ratio used to determine the organic contamination like polysaccharides.

Recommendation

This is recommended even in low technology laboratories for high thought sample preparation suitable
for various molecular analytical techniques.

It is advisable to screen different genotypes of where to obtain resistant genotypes.it is highly

recommended silica gel used as most common instrument for preserving animal cell tissues in the field
for future DNA extraction.

It is highly recommended to solidified Agarose gel after removal of the comb.