17.

- Electrophoresis of nucleic acids in agarose

gels. Electrophoretic isolation and
characterization of plasmid DNA
Carmen Alicia Padilla Peña, Jesús Diez Dapena, Emilia Martínez
Galisteo, José Antonio Bárcena Ruiz, Concepción García
Alfonso
Department of Biochemistry and Molecular Biology, Rabanales University Campus, Severo
Ochoa Building, 14071-Córdoba

SUMMARY

Agarose gel electrophoresis is one of the most commonly used to analyze

and characterize nucleic acids from different sources. Gels behave like a
molecular sieve and allow to separate charged molecules depending on
their size and shape. Thus, DNA molecules of different size will migrate
differently in an agarose gel electrophoresis. In addition, if molecular
weight markers (DNA fragments of known size) are applied in said
electrophoresis, the approximate size of the DNA under study can be
calculated. In this practice, it is intended that the student understand the
theoretical basis of this technique and its applications, carrying out the
electrophoretic separation in agarose gel of plasmid DNA that the same
student will isolate and purify from a bacterial culture. A large number of
methods have been developed for the purification of plasma DNA from
bacteria and all of them invariably involve three steps: growth of the carrier
bacterial strain, collection and lysis of bacteria and purification of plasma
DNA. The isolated DNA can then be analysed by agarose gel
electrophoresis.

Keywords: nucleic acids, electrophoresis, agarose gel, molecular weight

markers, plasmids.

Abbreviations used. DNA: ácido desoxirribonucleico; LB: Luria-Bertani;

SDS: dodecil sulfate sodium; TBE: Tris-borate-EDTA (ethylene
diamine
tetraacetic); UV: ultraviolet light.

1. INTRODUCTION AND OBJECTIVES

The term electrophoresis is used to describe the migration of a charged

particle under the influence of an electric field. Many biologically important
molecules (amino acids, peptides, proteins, nucleotides, nucleic acids...) have
ionizable groups and exist in solution as charged species, either as cations, or as
anions. These charged species are to be separated according to their charge
when a voltage is applied across the electrodes. There are many types of
electrophoresis, which fall into two fundamental categories:
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 -Electrophoresis of mobile front.
-Electrophoresis of zone.

Currently, only zone electrophoresis is used, in which the sample moves on a

solid support, such as filter paper, cellulose or gel (agarose, acrylamide...) and
the sample components migrate in the form of small bands, also called zones.

Gel electrophoresis is a widely used technique for separating molecules or

fragments of nucleic acid molecules. The most common materials for separating
nucleic acid molecules are polymers such as polyacrylamide or agarose. These
gels are placed in the electrophoresis basin, immersed in a buffer of pH around
8. In this way, DNA or RNA molecules subjected to electrophoresis will move to
the positive pole since at pH greater than 5 they have a negative charge. Gels
behave like a molecular sieve and allow to separate charged molecules
depending on their size and shape. So, DNA molecules of different sizes will
migrate differently in an electrophoresis gel. The distance travelled by each DNA
fragment will be inversely proportional to the logarithm of its molecular weight. It
is important to use markers of known size because they will allow us to calculate
the molecular weights of the test DNA samples.

In the case of agarose gels, ethidium bromide is added, a substance that is

sandwiched between the bases of DNA and is fluorescent when illuminated by
ultraviolet light. After electrophoresis, the gel is visualized with a UV light lamp,
and the bands corresponding to the applied DNA samples and molecular weight
markers will be seen.

The problem DNA to be analyzed by agarose gel electrophoresis in this

practice is plasmid DNA. Plasmids are small, circular extracromosomal DNA
molecules (between two and several hundred kilobases) found in many bacterial
species. They are characterized because they replicate independently of
bacterial chromosomal DNA, have their own replication origin, and are not
necessary for the overall viability of the cell, but may contain genes that contribute
to survival under special conditions, such as antibiotic resistance. A certain
bacterial cell may not have any plasmids or may host a variable number of
plasmids. Some kinds of plasmids possess the property known as "relaxed
replication", that is, they are present in the form of many copies per cell, which
greatly facilitates their isolation and purification.

A large number of methods have been developed for the purification of

Plasmid DNA of bacteria and all of them invariably involve three steps:
a) Growth of carrier bacterial strain in culture medium
b) Collection and lysis of bacteria
c) Purification of plasma DNA

The method used to obtain plasmid in this practice is called alkaline lysis and
is based on the selective denaturing of chromosomal DNA by NaOH alkalinization
and addition of SDS

2
(detergent) , under conditions in which the plasmid DNA remains close to its
native structure, mainly due to its small size and its circular and superenrolled
nature. By neutralizing the medium and adding a high concentration of salt
(potassium acetate), much of the protein is precipitated due to SDS treatment
and high salt concentration. It also precipitates chromosomal DNA, probably
because random reassociations occur between different regions of this DNA and
thus insoluble aggregates are formed. In contrast, it does not precipitate plasmid
DNA, which remains in the supernatant. This DNA can be analyzed and
visualized by agarose gel electrophoresis. In each of the streets of the gel where
the plasmid DNA is loaded, we will see three bands of different size that will
correspond to the three main forms with which it migrates the same depending
on its degree of winding. They are the circular, rolled and supercolluted forms
that migrate with less to higher speed, respectively, in an electrophoresis.

Finally, we will prepare a calibration line with the molecular weights of the
markers (phage fragments λdigested with HindIII) depending on the distance
travelled in the electrophoresis and we will interpolate the distances covered by
the different states of the plasmid’s winding to know its molecular size.

The fundamental objectives of this practice are two:

• Become familiar with methods of separation of nucleic acids by

electrophoresis in agarose gels.
• Isolation and purification of bacterial plasmids from carrier strains by using
a commercial "Kit".
• Determination of molecular sizes by agarose gel electrophoresis, making a
straight pattern with DNA fragments of known molecular weight.

2. LIST OF MATERIAL REQUIRED

2.1. Equipment

Desktop eppendorf centrifuge.

Microwave or hot plate.
Electrophoresis equipment (bucket, holder, comb, power supply).
UV light transilluminator.
Imaging and thermal photography equipment.

2.2. Material

Small flasks.
5 mL glass pipettes.
Test tubes.
Automatic micropipettes and disposable tips for them.
1.5 mL eppendorf tubes.

2.3. Reagents

3
 Culture medium LB.
Bacterial culture carrying plasmid.
Solution of cell resuspension.
Cell lysis solution (NaOH/SDS).
Neutralizing solution.
Small columns and solution "Quamtum Prep".
Washing solution.
Sterile water.
Electrophoresis buffer TBE (Tris-borate).
Agarosa.
Ethidium bromide (VERY DANGEROUS, WEAR GLOVES!!).
Loading buffer for samples.
Molecular weight markers.

3. PROTOCOL TO BE IMPLEMENTED

3.1. Preparation of plasmid carrier bacterial culture

3.1.1.Prepare culture flasks or 5 mL (Luria-Bertani) tubes, to which the

appropriate antibiotic is added when growing.

3.1.2.An individual bacterial colony is taken from a solid culture medium plate into
the liquid medium and incubated overnight at 37 ºC with stirring.
NOTE: Each group will prepare the crop for the next day’s students and use a
crop already prepared the day before. ¡ This section of the protocol will be
performed while agarose gel electrophoresis is performed!

3.2. Isolation and purification of plasmid DNA

3.2.1.1 1.5 ml of the bacteria culture is taken and placed in an eppendorf, then
centrifuged for 1 minute at 14000 rpm. The supernatant is removed, leaving the
sediment as dry as possible.

3.2.2.The bacterial sediment is resuspended in 200 µl of cell resuspension

solution using the pipette (the liquid is aspirated and expelled several times until
the sediment is resuspended).

3.2.3.250 µl lysis solution (NaOH/SDS) is added to the same tube. Mix the
contents of the tube by soft inversion about 10 times. The solution will appear
viscous and slightly clear if cell lysis has occurred.

3.2.4.250 µl neutralisation solution (potassium acetate) is then added. Mix the

contents of the tube by soft inversion about 10 times. A visible white
precipitate containing chromosomal DNA will appear along with the
proteins.

3.2.5.Centrifuge 5 min at 14000 rpm and prepare the column, placing it in a tube.

4
3.2.6.Transfer the supernatant to the column with the micropipette, avoiding
touching the sediment. In this supernatant is the DNA of the plasmid.

3.2.7.200 µl of "Quantum Prep" solution are added to each column. Mix well
using the pipette.

3.2.8.Centrifuge for 30 seconds at 14000 rpm.

3.2.9.The filtrate is removed by dumping the tube and placing the column back
into the tube.

3.2.10.500 µl of washing solution is added to the column and centrifuged

30 seconds at 14000 rpm.

3.2.11.Remove the filter and repeat the previous step.

3.2.12.Change the column to a clean eppendorf tube and add 100 µl elution
solution ( H2O).

3.2.13.Centrifuge 1 minute at 14000 rpm and the collected solution is the

preparation of isolated and purified plasmid DNA.

3.3. Preparation of agarose gel

3.3.1.Pour into a flask 30 ml of the TBE electrophoresis buffer and 0,21 g of

agarose (0,7 %).

3.3.2.Melt the agarose solution in a microwave or hot plate.

3.3.3.Allow the suspension to cool to approximately 50ºC.

3.3.4.Seal with adhesive tape the support where the agarose gel is to be poured
and place the comb that will serve to form the wells of the gel.

3.3.5.Once the agarose solution has reached 50ºC, add 2 µL of ethidium bromide
(10 mg/ml) and mix well. ¡ PUT GLOVES!!!

3.3.6.The agarose solution with ethidium bromide is poured into the previously
sealed support. Allow to polymerize for about 30 minutes.

3.4. Preparation of DNA samples

3.4.1.20 µl of the isolated plasma DNA is taken in the first part of the practice
and 5 µl of charge buffer is added.

3.5. Electrophoresis

5
3.5.1.Remove the adhesive tape with which the gel holder has been sealed and
place it in the electrophoresis bucket. EYE!: The wells should be near the cathode
(negative pole, black).

3.5.2.TBE electrophoresis buffer is added to cover the agarose gel well.

3.5.3.10 µl of the sample prepared in the previous section is applied in two wells.

3.5.4.10 µl molecular weight markers are applied in two of the wells.

3.5.5.Plug the electrophoresis cell and connect the electrodes to the power
supply. It is checked that it is well connected.

3.5.6.Program the source at about 100 volts and start running the electrophoresis
that will last about 30-45 minutes.

3.5.7.Once the electrophoresis is finished, the DNA fragments are visualized by

UV light and a photograph of the image is made.

4. EXPECTED RESULTS

The image obtained from the gel through UV viewing should be similar to the
following:
Markers Markers
weight DNA DNA weight
molecular Plasmídico Plasmídico molecular

Polo -

23 KB

9 KB
6 KB
4 KB

2.3 KB
2 KB

0.5 KB

Polo +

5. DISCUSSION AND COMMENTS

Once the practical part is finished, and making use of the image obtained from
the electrophoresis gel, the student will have to discuss the results obtained and
make the appropriate comments. To do this, you should pay attention to the
streets of the gel where you have applied plasma DNA samples. Three bands of
different intensity and size should be seen, corresponding to the three main forms
of plasmid DNA enrollment.

6
 The individual student must calculate the size, expressed in base pairs, of
each of these three bands, making use of a straight pattern that will have to be
constructed with molecular weight markers used in electrophoresis.

Each student will be given a sheet with the following image to make the straight
pattern and calculate the sizes of the different forms of plasmid DNA. In addition,
a series of questions will be included to check that the student has understood
the practice.
STRAIGHT

Distance
of
migration
(mm)

Logarithm of molecular weight (Kb)

6. ANNOTATED BIBLIOGRAPHY

Bio-Rad: Protocol that accompanies the "Quantum Prep Miniprep Kit".

Nelson DL, Cox MM (2001): "Principles of Biochemistry", 3rd ed. Editorial Omega
(Barcelona, Spain), pp 1119-1128.
Stryer L, Berg JM, Tymoczko JL (2003): "Biochemistry", 5th ed. Editorial Reverté
(Barcelona, Spain), pp 152-154.

7
ANNEX 1: MEANS, SOLUTIONS AND BIOLOGICAL MATERIAL USED

Culture medium LB

Culture medium LB
Liquid medium Solid medium
(g) (g)
“Bacto tryptone” 10 10
“Bacto yeast extract” 5 5
ClNa 10 10
Hagar - 15
Distilled water Up to 1 litre Up to 1 litre

Electrophoresis buffer TBE

1X TBE buffer solution

5.0 litres 1 liter
(g) (g)
“Tris base” 55,8 11,16
Boric acid 42,1 8,42
EDTA-Na2 4,9 0,98
Distilled water Up to 5 litres Up to 1 litre

8
Electrophoresis of nucleic acids in agarose gels. Isolation and characterization Plasmid
DNA electrophoresis

Guerrero Morrón Andrea Melissa

Muñoz Orozco Jorge David
Ospino Quevedo Dayana Carolina
Peña Medina Gisela María

Presentado a:
Yina Paola García Toscano

Universidad Metropolitana.

Bacteriología.

Biología Molecular

IV Semestre.

Barranquilla.

2023.
English.

Agarose gel electrophoresis is a widely used technique to analyze and characterize nucleic
acids, such as DNA. Agarose gels act as molecular sieves that separate charged molecules
based on their size and shape. This allows the migration of DNA fragments to be observed
depending on their size during electrophoresis. Furthermore, by using molecular weight
markers, it is possible to estimate the size of the DNA under study.
In this practice, plasmid DNA from bacteria is isolated and purified through a process that
involves the growth of the carrier bacteria, their collection, cell lysis, and purification of the
plasmid DNA. Subsequently, the plasmid DNA is analyzed by agarose gel electrophoresis.
Plasmid DNA is a circular DNA molecule that replicates independently of bacterial
chromosomal DNA and may contain genes of interest.
The plasmid DNA is separated on the agarose gel based on its shape and size, and a
molecular weight marker is used to estimate its size. The alkaline lysis technique is used for
the purification of plasmid DNA, and the gel is stained with ethidium bromide to visualize
the DNA bands.
The objectives of this internship include becoming familiar with the technique of agarose gel
electrophoresis, isolation and purification of plasmid DNA, and determining the molecular
size of DNA by electrophoresis.
The protocol details the steps necessary to prepare the bacterial culture, isolate and purify the
plasmid DNA, prepare the agarose gel, and load the DNA samples. Specific equipment and
reagents are used to carry out each step of the practice.
Español.

La electroforesis en gel de agarosa es una técnica ampliamente utilizada para analizar y

caracterizar ácidos nucleicos, como el ADN. Los geles de agarosa actúan como tamices
moleculares que separan moléculas cargadas según su tamaño y forma. Esto permite observar
la migración de fragmentos de ADN en función de su tamaño durante la electroforesis.
Además, al utilizar marcadores de peso molecular, es posible estimar el tamaño del ADN en
estudio.
En esta práctica, se aísla y purifica el ADN plasmídico de bacterias mediante un proceso que
involucra el crecimiento de las bacterias portadoras, su recolección, lisis celular y
purificación del ADN plasmídico. Posteriormente, se analiza el ADN plasmídico mediante
electroforesis en gel de agarosa.
El ADN plasmídico es una molécula circular de ADN que se replica de manera independiente
del ADN cromosómico bacteriano y puede contener genes de interés.
El ADN plasmídico se separa en el gel de agarosa en función de su forma y tamaño, y se
utiliza un marcador de peso molecular para estimar su tamaño. La técnica de lisis alcalina se
emplea para la purificación del ADN plasmídico, y el gel se tiñe con bromuro de etidio para
visualizar las bandas de ADN.
Los objetivos de esta práctica incluyen familiarizarse con la técnica de electroforesis en gel
de agarosa, aislamiento y purificación de ADN plasmídico, y determinar el tamaño molecular
del ADN mediante electroforesis.
El protocolo detalla los pasos necesarios para preparar el cultivo bacteriano, aislar y purificar
el ADN plasmídico, preparar el gel de agarosa y cargar las muestras de ADN. Se utilizan
equipos y reactivos específicos para llevar a cabo cada paso de la práctica.

Video.
https://youtu.be/1d1M-RhaGzA?si=JYO24c81JYvXs_ud.