Arabi 1

Name: Ahmad Abdulkadir Arabi (A00023244)

Instructor: Dr. Malachy Okeke

Lab Report 1C

Date: 4th June, 2022

Title:

Agarose gel electrophoresis of isolated human and virus DNA

Abstract:

The lab's final objective was to properly quantify the size of various DNA samples by inserting
them in wells in an agarose gel and passing a current through a charged chamber. The DNA
samples will migrate towards the positive charge as they pass through the gel. In an ideal world,
the DNA would travel and form a sequence from smallest to largest. Each band in the DNA
sample is predicted to have the same length (in base pairs) as the bands of known length in the
DNA size standard. The first band has around 6000 base pairs, the second has about 3500 base
pairs, and the third has about 1500 base pairs.

Introduction:

Gel electrophoresis is used to separate macromolecules like DNA or RNA by size or proteins by
charge. The pieces of DNA and RNA are inserted in agarose wells and an electrical charge is
applied, forcing the negatively charged molecules to the positive side. The smaller the molecule,
the less resistance it will encounter when it hits the pores of the gel, and hence the greater the
distance it will travel.

When DNA and RNA molecules are separated using the gel electrophoresis technology, DNA
fragments separate according to size when subjected to an electric field through a gel matrix, a
jam-like porous substance. Because DNA is negatively charged, it travels through the gel toward
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the positive pole when subjected to an electrical field along these lines. The gel matrix functions
as a sieve through which DNA molecules travel; larger pieces have a harder time passing
through the pores of the gel, and so migrate through the gel more slowly than smaller DNA
fragments. This means that once the gels have been electrophoresed or "ran" for a certain amount
of time, pieces of different sizes are separated because they travelled at different speeds through
the gel (GANN, JAMES D. WATSON ALEXANDER BAKER et al. 1966).

The separation of DNA fragments for DNA fingerprinting to examine crime scenes, to inspect
results of polymerase chain reaction, to analyze genes qualities related to a specific illness, and
in DNA profiling for taxonomy study to separate various species are just some of the
applications of gel electrophoresis. In paternity testing using DNA fingerprinting, in the study of
protein structure and capacity, in the investigation of antibiotics, in smearing procedures for
macromolecule investigation, and in the investigation of developmental connections by breaking
down hereditary similarity among populations or species (Cheriyedath 2018).

In this experiment, Plasmid puC19 was digested with BamH1, EcoR1, AvaI we run gel
electrophoresis to separate human and virus DNA bands respectively based on their sizes.

Materials:

Flask

DNA (Stock and serially diluted) from Part A

1kb DNA Ladder

TE Buffer

1 X TAE buffer (made from 50 X TAE)

Ethidium Bromide (10mg/ml)

6 X Loading buffer

Nanodrop Spectrophotometer

P10, P20 and P100 Pipet

Gel casts
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Microwave

Gel box

Gel Combs

Agarose

Masking Tape

Gel documentation system

Plastic container for carrying the gel

Pipet tips

Method:

A 1.2 percent gel for human DNA was prepared in 120 ml of 1 X TAE buffer and a 0.6 percent
gel for viral DNA too in 120 ml of 1 X TAE buffer, respectively. The gel was assembled in the
following manner: Then, the gel was solubilized and heated for 1 to 3 minutes. 5µl of EtBr was
poured into the solubilized gel and was stirred well. Then the gel was poured into the gel cast
and was let to aside for 30 minutes to harden. In duplicate, four Eppendorf tubes were marked
with of 10-1 to 10-4. Each Eppendorf tube was filled with 18µl of distilled water. 2µl each of the
extracted human and viral DNA (isolated from component B). To the first pair of tubes (10-1),
2µl of the extracted human and viral DNA was added respectively (isolated from part B of the
lab session), and a tenfold serial dilution was repeated until 10-4 Pipet 4µl of stock DNA (human
and viral DNA) and 10µl of serially diluted DNA (from part B) is freshed into new Eppendorf
tubes, labeling them as undiluted, 10-1, and 10-2, respectively. In a tube containing 4µl of pure
stock DNA, 6µl of distilled water was added, followed by 2µl of loading buffer.  2µl of loading
buffer was added to tubes containing diluted DNA (1: 10 and 1:100 dilutions (10-1 and 10-2) and
thoroughly mixed pipetting up and down. The gel cast was placed in the electrophoretic chamber
(gel box). With the comb and the masking table was removed. In 1 X TAE running buffer, gently
submerge the gel. Fill gel 1 with the following samples: Lane 1 (4µl of 1 kb ladder), Lane 2
(10µl of dH2O), Lane 3 (10µl of undiluted human DNA), and Lane 4 (10µl of 1:10 diluted
human DNA), and Lane 5 (10µl of 1:100 diluted human DNA). Lane 1 (4µl 1 kb ladder), Lane 2
(10µl dH2O), and Lane 3 (10µl undiluted viral DNA), Lane 4 (10µl of 1:10 diluted virus DNA),
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and Lane 5 (10µl of l: 100 diluted plasmid DNA) were used for the second gel. The electrodes
were connected to the power source and the lid of the gel box was put on the gel box (RED to
RED and BLACK to BLACK). The gel was run at 80V for 80 minutes, or until the front dye had
migrated to roughly two-thirds of the length of the gel. After that, the power was switched off,
the lid from the gel box was removed, and the gel (in the gel cast) was carefully placed in the UV
box, with a photo of your gel taken.

Results:

Fig 1: Agarose gel electrophoresis of isolated human DNA

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Fig 2: Agarose gel electrophoresis of isolated virus DNA

Discussion:
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The estimated length in base pairs of each band in the DNA sample aove is based on how they
line up with bands of known length is the DNA size standard. The top band is approximately
6000 base pairs, the second is approximately 3500 base pairs and the third is approximately 1500
base pairs that had been ran on the gel.

Reference:
Cheriyedath, Susha M. S. 2018. “What Does Gel Electrophoresis Involve?” Last Updated: .

Retrieved November 7, 2019 (https://www.news-medical.net/life-sciences/What-Does-

Gel-Electrophoresis-Involve.aspx).

GANN, JAMES D. WATSON ALEXANDERBAKER, TANIA A., MICHAEL LEVINE,

STEPHEN P. BELL, and RICHARD LOSICK. 1966. Molecular Biology of the Gene

James D. Watson. Vol. 16. SEVENTH ED. edited by J. Inglis, A. Gann, J. Argentine,

and K. Janssen. New York: C OLD SPRING HARBOR L ABORATORY P RESS Cold

Spring Harbor.