MOHAWK COLLEGE OF APPLIED ARTS AND TECHNOLOGY

CHEMICAL AND ENVIRONMENTAL TECHNOLOGY DEPARTMENT

Lab Report

ROOM NO: FE E309

EXPERIMENT NO : # 2

TITLE : Gel Electrophoresis of DNA

Submitted by : Fatima Alhubaishi ………………………….

Class : BIOL 10016 lab.

Partners : 1) Edvair Moreira

2) Rachel Joseph
3) Claudia Leonce

Instructor : Farag Soliman

Date lab performed : June 3, 2021

Date of submission :

FENNELL CAMPUS
HAMILTON, ONTARIO
Purpose:

To carry out agarose gel electrophoresis of DNA.

Procedure:
1) Prepare the Agarose gel:
 Add 0.4g of Agarose powder to a 50 ml buffer solution in a 200 beaker
 Heat the Agarose solution in a microwave on a medium low heat for 2-3 minutes
until it turns perfectly clear
 Leave the beaker to cool down. When the beaker is hot enough to handle, add
0.5 ul of 10 mg/ml Ethidium Bromide to the Agarose solution.
2) Cast the gel after setting up the Electrophoresis chamber by making any necessary
adjustments and choosing the appropriate comb for the setup.

Chamber Adjustment
 Adjust the leveling feet of chamber by tightening, and then make sure that the
work surface is level with a built in bull’s eye.
 After inserting the rubber pouring dams, put the tray into the chamber.

Comb section:
 Using the depth adjustment screws attach the selected comb with the
required number of wells to comb holder.
 Use the comb to position the comb frame so that it is parallel to the rubber
pouring dam at the beginning of the screed and then remove the depth level.

Gel casting:
 Pour the liquid gel and let it dry for a total of 30 minutes until it sets.
 Carefully remove the comb holder and the rubber end blocks.
 Adjust the gel tray so the sample well is closer to the cathode ( negative
electrode, black) rather than the anode ( positive electrode, red).
 Pour the buffer solution until the top surface of the 1mm gel is completely
submerged.
3) Load the prepared DNA sample after mixing 50 α 1 Lambda DNA digested with 10 α 1
use proper pipetting techniques to load with dye into 1.5 ml microcentrifuge tube.
4) Check the polarity of the leads after attaching the chamber to the power supply.
5) Run the gel at 140 volts for 35 minutes until the ethidium bromide dye settles to the
bottom of the gel.
6) Turn off the power and carefully remove the gel.
7) After placing the gel in the UV box, view the fluorescent DNA band under UV light and
use the Bio-Rad Gel Doc system to take pictures of the gel.

Materials:

Agarose gel, Ethidium Bromide, Electrophoresis Chamber, Casting tray and Comb, TBE buffer solution,
Micropipette with tips, Beaker, U.V Light box.
Observation:
After conducting the experiment it can be observed that the DNA molecules move from the negative end
(anode) of the electrophoresis chamber to the positive end ( cathode). Instead the smaller band travels
farther, while the larger band covers the shorter distance in comparison.

Amount of the DNA in the wells

Volume (µL) Mass (µg)

4 2

Determine of Lambda DNA mass in 8 fragments (Bands):

Size of Mass added to Mass of DNA in each

% of Lambda
Band Band well Band
DNA
(bp) (µg) (µg)
1 23130 48 2 0.95
2 9416 19 2 0.39
3 6557 14 2 0.27
4 4361 9 2 0.18
5 2322 5 2 0.10
6 2027 4 2 0.80
7 564 1 2 0.02
8 125 0.3 2 0.01

Calculations:

For Band 2:
 % of Lambda DNA in a fragment = 23130
48502 x 100
= 48%
  Lambda DNA mass (µg) in the fragment = 2 µg x 48
100

= 0.96 µg

Graphs/ Diagrams
Discussions:

The DNA molecule is negatively charged in its composition for the presence of the nucleotides and is
loaded towards the negative end of the electrophoretic chamber. Therefore execute DNA samples in the
electrophoresis chamber, moves from a negative end (anode) to the positive ( cathode) for a strong
traction voltage. DNA bands are separated according to their molecular weight since the threads with
minimum molecular weight or the minimum are not easier. A pair of base that move faster than the
DNA chain with the highest molecular weight.

Conclusion:

Therefore, gel electrophoresis is a useful technique to separate DNA fragments from other large
molecules (such as RNA and proteins) based on size and charge. If a strand of DNA contains more
restriction enzymes, the strand of DNA will be cut into smaller fragments and moved further to the
positive side during the gel electrophoresis test. The smaller the size of band the further it travels.

Post lab questions:

1- Assume an unknown sample of DNA was run in a separate lane, and was positioned, adjacent to
the 4361 bp band of the Lambda/HindIII lane, with the same brightness; What information can
be determined about the unknown DNA from the electrophoresis results? Be specific. Give an
application where this information would be important.

Since the unknown samples have the identical brightness it also have the same number
or the same molecular weight. Lambda/ Hind III i.e. 4361 bp. Thus, its position will correspond
to the Lambda/ Hind III band. This information would be most vital when conducting research
reference to the employment of the said enzyme.

2) Why is the DNA sample predigested with Hind III before the electrophoresis procedure? How would
the electrophoresis fingerprint look, if the DNA had not been predigested?

The DNA sample is predigested with Hind III so it may be separated into bands of various size will be
identified in keeping with no. of base pairs present in them. If the DNA had not been predigested only
one non-segregated band would are visible on exposure to the U.V light.

3) Explain how Ethidium Bromide interacts with the DNA molecule. Be specific.

Ethidium Bromide the double stranded DNA changes the charge conformation also because the relative
molecular mass of the DNA. Thus upon illumination with U.V light the banding pattern becomes visible
to the eyes.
Reference:

Sigmon , Larcom L (1996) The effect of ethidium bromide on the mobility of DNA fragments in agarose
gel electrophoresis. Electrophoresis 17(10) :1524-7.doi: 10.1002 / t elps.1150171003.
https://pubmed.ncbi.nlm.nih.gov/8957173/