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Experiment 4

This document describes an experiment on separating DNA fragments by size using agarose gel electrophoresis. [1] The student prepared a 0.7% agarose gel and loaded DNA samples extracted from bananas and strawberries along with a 1kb ladder standard. [2] After electrophoresis, bands were visible between 250-1000bp for one strawberry sample by comparing it to the ladder. [3] Other samples may not have shown bands due to errors like bubbles or incorrect loading.

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35 views

Experiment 4

This document describes an experiment on separating DNA fragments by size using agarose gel electrophoresis. [1] The student prepared a 0.7% agarose gel and loaded DNA samples extracted from bananas and strawberries along with a 1kb ladder standard. [2] After electrophoresis, bands were visible between 250-1000bp for one strawberry sample by comparing it to the ladder. [3] Other samples may not have shown bands due to errors like bubbles or incorrect loading.

Uploaded by

imenmezhoud1122
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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HALIC UNIVERSITY

FACULTY OF ARTS AND SCIENCES


DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS

MBG204(2)
GENETICS LABORATORY

INSTRUCTOR
Dr. Allison Pinar Eronat

EXPERIMENT 4:
Agarose gel electrophoresis

Experiment Date:30/03/2022

Name of the Student:


YMEN MAZHOUD

Submission Date of the Report: 06/04/2022

1. Aim of the experiment:


This experiment aims to separate DNA fragments according to their size using the
agarose gel electrophoresis technique and to compare them to a standard “ladder”
sample.
2. Introduction:
Electrophoresis is one of the several techniques that can be used in laboratories to
isolate DNA and RNA fragments. This isolation technique is based on the size
and charge of the particles of nucleic acid. The gel contains pores that act as
filters for the molecules passing through them. In order to be able to estimate the
size of the DNA fragments, standards called “ladder” are also separated on the
same gel. The results of the standard and the studied sample are then compared. In
agarose gel electrophoresis, a circuit is generated at the two ends of the gel.
Knowing that the DNA is negatively charged, it can only move from the negative
pole to the positive pole. Therefore, both the size and charge of each particle
decide how far it travels along the gel.
In addition to the gel, the cassette containing the gel must be immersed in a
suitable buffer solution. Electrophoresis is a time-efficient technique used for
DNA analysis. (figure 1) demonstrates a basic gel electrophoresis setup used in
laboratories.

Figure 1. Gel agarose setup. [1]


Electrophoresis can be run vertically or horizontally; agarose gel electrophoresis
is run horizontally whereas polyacrylamide gel runs vertically. Polyacrylamide
gel electrophoresis is more accurate compared with agarose gel. Its precision can
even detect the difference in one base pair.
3. Materials:
Equipment:
- Electrophoresis tray
- Comb
- Erlenmeyer flask
- Graduated cylinder
- Cellophane
- Micropipette
- Parafilm
- Watch glass
- Spatula

Devices:
- Precision balance
- Microwave
- UV illuminator
- Electrophoresis unit
- Electric source

Chemicals:
- 0.7% agarose
- Ethidium Bromide (EtBr)
- 50 ml 1x TAE buffer
- 1kb ladder
- Loading dye

Organic material:
- Strawberry DNA
- Banana DNA

4. Experimental procedure:

i. Preparation of agarose gel (0.7%, 50mL):


- To prepare a 0.7% agarose solution, 0.35g of agarose is weighed and added to an
Erlenmeyer flask.
- 50ml of 1x TAE buffer solution is measured using a graduated cylinder and added to
the agarose powder.
- Using a cellophane film, the Erlenmeyer flask is covered to minimize water vapor
escape during the heating process.
- Two small holes are made in the parafilm.
- The flask is then placed inside the microwave oven for two minutes, at 850W.
- Once the mixture is homogeneous, it is left to cool down (50-60 degree celsius)
- 1.5µl of EtBr is added to the Erlenmeyer flask and mixed gently. High precautions
must be considered when using this chemical.
ii. Agarose gel electrophoresis procedure:
- A gel cassette and two plastic barriers are assembled in a way that no potential
leakage of the gel might occur.
- A comb is placed at the end of the tray. This comb is to be removed later to make
wells where the samples are to be pipetted.
- The gel is poured into the tray and left to solidify. Once solidified, the two plastic
barriers are removed carefully.
- The cassette is then placed inside a buffer tank with two electrodes. TAE buffer is
poured into the tank until the cassette is fully submerged.
- The wells formed after the removal of the comb will be used for sample placement.
- In the first well, 3µl of 1Kb ladder solution is loaded.
- 1 µl of loading dye and 5 µl of DNA are diluted on a parafilm. The dilute mixture is
loaded into the next well. No air bubbles must be inserted while mixing the two
substances.
- The same step is repeated for all the available samples using the same volume ratio “1
µl loading dye: 5 µl DNA sample”.
- The agarose gel is run at 200V for ten to fifteen minutes.
- The obtained gel is observed under UV light using the UV illuminator. The results of
each sample must be compared to the standard from the first well.

5. Results:
After running the gel at 200V for 10 minutes, the following result was obtained (figure
3). We can observe different stripes situated at similar distances in well 1, 7 and 8.

The remaining wells do not have any visible results. The control M is to be used to
identify the DNA fragments size.

Figure 3. Agarose gel electrophoresis of DNA samples extracted from bananas and
strawberries. [2]
6. Discussion:
In this experiment, we loaded DNA samples obtained from the experiment “DNA
isolation” into a gel electrophoresis plate. After 10 minutes, the cassette was observed
under UV light and the picture from (figure 3) was obtained.
In the figure, the control M is used to estimate the size of the DNA fragments. In well 1,
the DNA fragments are not clear which makes it harder to identify. However, in well 7, a
very clear sample is obtained. After comparing it with the ladder (figure 4), the DNA
fragments range in size between 250 and 1000bp.
The reason why no DNA is observed in the remaining wells can be explained due to so
many experimental errors. First, air bubbles might have been inserted along with each of
these samples, decreasing the volume to less than 6 µl. Another reason is that the sample
might have been loaded the wrong way into each well. The sample should not be injected
into the gel nor loaded at the surface. It must be loaded near the base of the well without
touching the gel.

Figure 4. Gene ruler 1kb DNA ladder []


7. References:
[1] https://www.sciencedirect.com/topics/pharmacology-toxicology-and-
pharmaceutical-science/agarose-gel-electrophoresis accessed on 05/04/2023

[2] Cebeci, A., Genetics laboratory: results of experiment 4; Agarose gel


electrophoresis, 2023, Halic University, Faculty of Sciences and Fine Arts.

[3] Document Connect (thermofisher.com) accessed on 05/04/2023

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