Title of experiment

DNA Extraction and Quantification-Quality Analysis

Name and Surname: Tuğçe ÇİMEN

Student ID :210104003

Department :Molecular Biology and Genetics

Date of experiment : 18.11.2014 - 25.11.2014

Abstract

In our experiment,the extraction of genomic DNA that purification of the desired will be
done.Also,quality and quantity analysis of this DNA will be done.DNA extraction is a kind
of seperation,purification method.We learn to obtain DNA by help of destroying structures
such as the cell membrane or cell wall with chemicals or solutions in the experiment.And
main purpose is observing the DNA extraction and identification of nucleic acid fragments
with fractionation by gel electrophoresis method with agorose gel in our experiment.The
reason we choose this method is the proteins,RNA,polysaccharides,essential oils,phenols etc.
are minimal at DNA obtained from.Also we learn determining the purity and quantity of
obtained DNA by help of gel electrophoresis and UV spectroscopic measurements.Then we
can have some informations about DNA and its structure.And we comprehend that
DNAextraction method is used in PCR’s or sequencing researches by the experiment.

Introduction

As mentioned in the abstarct section,DNA definition is defined us with its structure and
attributes in the experiment.And the used methods for analysis of DNA are electrophoresis
method with agorose gel and UV spectroscopic method in the DNA extraction and analysis
are presented by help of the experiment.Also the materials and techniques used in the
experimental stages are familiarized and also we comprehend the aims and process of
experiment, in this section.

A-What is the DNA?

DNA or deoxyribonucleic acid for all living organisms and some viruses is a nucleic acid that
includes the genetic informations about liveliness.
The basic mission of DNA is the genetic material that stores instructions for a long time.DNA
forms from two long polymers that has simple units called nucleotide.The constituents of
DNA connect with ester bonds each other and consist of sugars and phosphote groups.In
eukaryotic organisms DNA takes place in the cell nucleus but in prokaryotes DNA takes place
in the cytoplasm of cell.

-Chargaff’s Rules-

Chargaff is a scientist.And he showed the relative ratios of the four bases were not random in
DNA.By the experiment,it is known;the number of Adenin(A) equaled the number of
Thymine (T) and the number of Guanine (G) equaled the number of Cytosine (C) in
DNA.So,the relationship is named Chargaff’s rules : A=T and G=C.

B-What is the genomic DNA extraction and How to genomic DNA extract?

Genomic DNA is sample DNA that contains all the genes in a cell.It has a large and complex
structure.It appears viscous in aqueous environment.The several causes of genomic DNA
extraction are:

*Profiling and fingerpriting (blood / sperm sample for comparison and forensic process)

*The first step in the process of gene cloning.

*Characterization and identification (for example transgenic controls modification of

animals).

*To investigate the regulation of gene expression.

*Research genetic disorders or diseases.

DNA extraction is a kind of purification and analysis.Due to this method the DNA
decomposes from protein, fat, RNA and other contaminants. First samples are obtained from
a cell to perform this process.Lysis solution will be prepared and DNA is removed protein
and nuclear envelopes.After precipitation process of DNA is done with the help salt solution.
Consantre DNA obtained after the centrifuge process.Then continues the precipitation with
isopropanol.After washing with distilled water and drying, the obtained DNA starts the
measurements.Start the quality and quantity analysis of the obtained genomic DNA.There are
different methods; flometric(slot blot method,microplac reader) or spectrophotometric (PCR
or spectrophotometer) methods.
The amount of contamination is determined by observing the measurements of the rate of
quantity and quality analysis.Method that we used for is the spectrophotometric method.The
equipment that we used is Scientific NanoDrop Spectrophotometer.And finally the observing
should be for visualize of DNA.There severare for this:

- spectral methods

-electrophoretic methods (agoros gel, pulsed field gel, capillary)

We used the agorose gel electrophoresis method for visualing DNA movements in the
experiment.

1-How to measure absorbance with Scientific NanoDrop Spectrophotometer?

Absorbance mesurement process is done with spectrophotometer,to measure the concentration

and purity of DNA within the fluid environment that we solve the DNA. We use a UV
spectrophotometer for the determination.Because the DNA by which the solution is the same
as the amount of DNA in the sample absorbed UV amount.

The ratio of 260/280

Nucleic acids at 260 nm, 280 nm for protein gives peaks.Pure DNA example, 260 and 280
nm absorbance ratio A260nm / A280nm = 1.8.According to this value indicates the efficiency
of the DNA sample in our hands.The value is directly proportional to the proximity of the
DNA yield.

The ratio of 260/230

This rate is a secondary method for the measurement of DNA purity.260/230 value for pure
nucleic acids is greater than 260/280.The value of this ratio is usually between 2.0 to 2.2.

So the UV absorbance of DNA used to determine purity and contamination.There are

certain wavelength of absorbance ratio in.This is done by measuring of the ratio Scientific
NanoDrop Spectrophotometer.To make measurements in the dry pit of NanoDrop,we put 2
microliters of DNA from our sample.We take measures to cover screw closing and press
button.We note the value of the ratio obtained:the ratios of 260/280,260/230 and
ng/microlitre.
figure 1. Scientific NanoDrop Spectrophotometer figure 2.example result graph

**References website for pictures:http://ulbread2.tistory.com/entry/Nanodrop-2

2- AgaroseGel Electrophoresis

Electrophoresis is a method of charged molecules containing liquid and in applied electrical

field environment for measuring the movement of the molecules.To be able to observe the
migration of particle types are used for the electrophoresis extraction of DNA .Identification
of nucleic acid fragments and is the most common method used for the purification.we will
use the method that is agarose gel electrophoresis. This method within a matrix of agoros gel
separators electric field exposure on dna fragments is a method of sorting out according to
size.This process consists of four stages:

- Preparation of the agarose gel:Agoros powder is mixed with prepared buffer solution.It is
heated in microwave oven until dissolved.After electrophoresis to see the DNA is mixed
ethidium bromide is added and cooled.

- Electrical assembly and preparation mechanism:The mechanism is prepared with

electrical chamber,electrical leads,gel casting trays and gel tank.

- The execution of the prepared gel: Samples are loaded to agros gel wells.Voltage adjusted,
the execution is carried out over a period of time.

- Gel visualized:Opening gel transimullator and conducted in DNA of ultraviolet light UV

are observed.
*Agorose

Agarose is a natural colloid derived from seaweed.It is very delicate and easily perishable
handling.Agarose gel has a large pore diameter.And used for the separation of larger
molecules from the ground at 200 kDa

Figure 3. Agarose’s structre,pores and powder forms

**References website:http://openwetware.org/wiki/BE.109:DNA_engineering/Agarose_gel_electrophoresis

3-Used Chemicals In The Experiment

tris: The buffering agent.

EDTA: by inhibiting the activity of DNase, DNA keeps stable.

NaCl: breaks through the cell wall proteins from DNA.

SDS: an ionic detergent, is involved in the destruction of membranee.

chloroform: Provides the breakdown of proteins.

isoamiyl alcohol: it stabilizes the intermediate phase, preventing the formation of bubbles.

ethanol: DNA used for washing and as a solvent.

isopropanol: used in the deposition process.

glycerol: provides a smooth progress by providing examples of crashing into the well.

bromophenol blue: Used to see what the walking process and the deposition process.

Ethidium bromide:.ethidium bromide is used in the preparation of agarose gel. İt is entering

the bonds between the DNA results show the effect of the fluorescent under UV light DNA
makes clear.
Materials and Methodes

1-)First Week Procedures

-First one leaves are cut into small pieces with a scalpel.

-We put the leaves pieces into mortar.

-Buffer solution is prepared to pour mortar.

-We prepared buffer solution with ; Nuclei lysis buffer (CTAB,EDTA and NaCl)

Sorbitol (buffer)

%5 Sarkosyl(detergent)

Sodium Bisulfite (salt)

-Added 10 ml of the buffer solution into the mortar with pipette.

- We mix to crush for the cell lysis.

- After the crushing process, we put 750 microliters of sample into the eppendorf tube with
pipette.

-The eppendorf tube is heated at 65ₒC in incubator.

- We are adding chloroform/isoamyl alcohol to the Eppendorf tube to remove fats.

- Eppendorf tube is vortexed for the separation of the phase for 20 seconds.

-After we centrifuge the tube for phase seperation and spin at 10,000 rpm for 5 minutes.

- Paying attention to the denser portions of the underlying, overlying liquid is taken and
transferred to a new eppendorf tube with pipette.

- After isopropanol was added to the precipitation of DNA with a new eppendorf tube.

- DNA is washed with 500 mL ethanol.

-It is centrifuged again to precipitate down the DNA.

- To fly the remains of liquid and ethanol the heating and drying processes are done.

-Distillation water is added and waited for resolving of DNA

- Then, Scientific NanoDrop Spectrophotometer is used for determining the DNA purity and
concentration at the appropriate wavelength.

- Firstly, nanodrop pit is cleaned and dried,then 2 microliters of the sample is taken and
placed in pit.

-Finally,amounts the ratio of 260/280,260/230 and ng/microliter are measured and recorded.

2-)Second Week Procedures

-Initially,the 3 gr Agorose is resolved in 300 ml buffer solution of %1.

-The solution is heated for 10 minutes, after that it is cooled for 20 minutes.

-After cooling 2 microliter of ethidium bromide is added and mixed.

-We prepare the mechanism by placing the gel combs into casting tray.

-And the agorose gel solution is poured intocasting tray,slowly.

-When the mechanism comes ready to install,we remove the gel combs and tapes than we put
the casting tray into the gel tank.

-And for samples we prepare loading buffer with using bromophenol blue and glycerol.Then
we add 5 microliter of loading buffer into the each samples.

-Then, we take 60 microliter of DNA samples and we load into the appropriate gel well with
pipette.

-And we have to be careful for loading without puncture to well.

-We are loading the ladder sample of 500 bp for comparison to the beginning of each row .

-Finally for runnig gel, electrical mechanism is prepared.

-Electric leadss are connected the correct direction of flow from negative to positive.We
place the mechanism in the electrophoresis chamber,connect the electrical leads to the power
supply.

-Then,power supply is worked with 95 electric flow for 45 minutes.- Finally The walk of
DNA is visualized on the ultraviolet transilluminator by DNA ladder standarts.
-Materials -

*Leaves Fragments

*Scalpel

*Mortar

*Eppendorf Tube

*Vortex

*Centrifuge Machine

*Micropipette

*Spatula

*Beaker

*Falcon Tube

*Incubator

*Nanodrop Spectrophotometer

*Power Supply

*Gel Tank

*Electrical Leads

*Casting Tray

*Gel Combs

*Ultra-violet Transilluminator

-Chemical Materials-

*Distillation Water

*Agorose

*Ethidium Bromide
*Bromophenol Blue

*Glycerol

*Chloroform/Isoamyl Alcohol

*Ethanol

*Isopropanol

*Nuclei Lysis Buffer(CTAB,EDTA,NaCl)

Results

1-The pictures of the results of laboratory steps

Figure 1.

We centrifuge to seperate the sample of DNA for disolving.

Also,we centrifuge the tube for phase seperation,spin at 10,000 rpm for 5 minutes.

So,we can observe the phases seperation in the eppendrof tube.

Figure 2. Figure 3.

By the figure 2 and figur 3 we can say that after we added isopropanol to the precipitation of
DNA with a new eppendorf tube.We washed DNA with 500 mL ethanol.It is centrifuged
again to precipitate down the DNA.Thus we can observe the precipitate of DNA at the bottom
of the tube.

2-The result of nanodrop spectrophotometer amounts

-My amounts of my DNA sample;

the ratios of 260/280: 2,12

the ratios of 260/230 :2,42

concentration ng/microlitre: 1352

If the amount of DNA is 1.8 to converge and efficiently DNA was obtained.
3-The visualized of gel

Figure 4.the ultraviolet photograph of DNA examples

**pictures references from our own photopgraphs in laboratory(figures of 1,2,3,4)

Discussion

We could explain a few examples that to be considered when making the genomic DNA
extraction. Ethidium bromide is a dye that works with ultraviolet rays.Experiment to be true,
during the addition of the dye should not be any air bubbles and care should be taken not to
damage of ultraviolet rays. while the gel voltage of care should be taken dissolve the gel.
When analyzed the image obtained as a result we see that isolation is not sufficient the
walk.There may be several reasons. it becomes difficult to walk because of the high
concentration gel, a corruption of slow walking balance. if we would like to view the DNA,
do not apply more ultraviolet rays.Because dna may be degraded, and may not run in the gel.
We have to pay attention to the air bubbles when the gel.Because if DNA cannot move in
space.We have achieved the top in fine white line agoros gel obtained from DNA
represents.There is no line during my group, because we've obtained the dna rna is
achieved.Wide white spaces left at the bottom of the resulting shows RNAs. By looking
NanoDrop spectrophotometer, the value in the efficiency and robustness of DNA are be
estimated. for example, my example is my value of 2.12.Normally, the value of the dna is
pure and efficient 1.8.Mine is not a very close value.That's why my DNA might be
broken.This may be the reason of the absence of the image.My experiments in the fault, my
sample into the bottom of the very tip of the straw back space when loading the gel cavity is
to suppress. After installation I had to remove my hand from pipette by without releasing the
button of pipette. To do this, my sample was dissolved and other gel-cell overflowed. may
make it difficult to walk to other space overflow.And I wouldn't be carrying space that occurs
in the image caused by mine sample. Thus,these images by examining dna we can observe
their work and technical operations in the experiment.

References

*From books:

-Molecular Biology of the Gene,James D. Watson,5th ed.,2004,ISBN 0-8053-4635-X.

-Gene cloning and DNA analysis:an introduction,T.A. BROWN,6th ed,ISBN 9781405181730

-Cell and tissue culture:laboratory procedures in biotechnology edited by Alan Doyle,J. Bryan
Griffiths,1998,ISBN 0 471 98255-5.

-Gen Klonlama ve DNA Analizi:Giriş,Nazan Yılmaz,translating from 5th

edition,Brown,T.A.,2009,ISBN 978-605-395-234-3.