BIOLOGICAL INSTRUMENTATION

PRACTICAL 3
DNA and Protein Gel Electrophoresis

DATE: 4/1/2021
Introduction for DNA Gel Electrophoresis
DNA Gel electrophoresis is a laboratory method used to separate DNA fragments according to
their molecular sizes. DNA samples are loaded into wells at one end of a gel, and an electric
current is applied to pull them through the gel. DNA fragments are negatively charged, so they
move towards the positive electrode.

Objectives
- To understand the principle of the DNA gel electrophoresis
- To learn and understand the technique of DNA gel electrophoresis

Materials
1. 10 µl max volume micropipette
2. 6 premixed DNA sample and loading buffer
3. DNA ladder (1kb or 100bp)
4. Prepared agarose gel
5. Electrophoresis buffer (TAE OR TBE)
6. Horizontal gel electrophoresis: gel casting platform, gel combs, DC power
Supply
Method
Discussions

1. Based on the image above:

a) State which sample has the shortest DNA size.
Sample B
b) State which sample has the longest DNA size.
Sample A
c) Identify which sample contains a 1kb size of DNA.
Sample A

2. State the function of agarose gel, gel combs, power supply, marker and
ethidium bromide.
Agarose gel is to separate the DNA fragments according to their sizes. Gel combs used to create
the wells in gels for electrophoresis, a technique that uses the electrical charges of molecules to
separate them by their length. Turning on the power supply sets up the electric field. Marker is a
set of standards that are used to identify the approximate size of a molecule run on a gel during
electrophoresis. Ethidium bromide is a molecule commonly used to visualize DNA in agarose gel
electrophoresis experiments.
3. Explain the principles of DNA migration in this experiment.
DNA gel electrophoresis separates DNA fragments according to their sizes. DNA fragments are
size separated by the aid of an electric field where negatively charged molecules migrate toward
anode (positive) pole. The migration flow is determined solely by the molecular weight where
small weight molecules migrate faster than larger ones.
4. What would happen if we set the voltage higher than 90 V? Provide your reason(s).
The higher the voltage, the faster the DNA will travel through the gel. However, voltages that are
too high can possibly melt the gel or cause smearing or distortion of DNA bands.

References
https://www.khanacademy.org/science/ap-biology/gene-expression-and-
regulation/biotechnology/a/gel-electrophoresis
https://en.wikipedia.org/wiki/Molecular-weight_size_marker
https://biologydictionary.net/gel-electrophoresis/
Introduction for Protein Gel Electrophoresis
SDS-PAGE is an analytical technique to separate proteins according to their molecular weight
based on their rate of migration through the gel matrix. SDS is a detergent with a strong protein-
denaturing effect that disrupt the protein and unfold the proteins into linear chains. There are two
types of buffer systems can be used, which are continuous buffer systems that use the same
buffer (at constant pH) and discontinuous systems that use a gel separated into two sections.
Objectives
- To understand the principle of protein gel electrophoresis
- To learn and understand the technique of protein gel electrophoresis
Methods
Discussions
1. Find Rf of the protein ladder using below formula:

Gel length=6.6cm
Protein ladder Log (MW) Length migrated Rf
Molecular weight (cm)
(MW) in kDa
245 2.39 0.6 0.09
180 2.26 0.7 0.11
140 2.15 0.9 0.14
100 2.00 1.3 0.20
75 1.88 1.6 0.24
60 1.78 2.0 0.30
45 1.65 2.7 0.41
35 1.54 3.5 0.53

2. Plot graph log (MW) vs Rf of the protein ladder using the data measured and
calculated in previous table.

Chart Title
3

2.5

1.5

0.5

0
0 0.1 0.2 0.3 0.4 0.5 0.6
 3. Then, determine the size of protein fragments in Sample A and Sample B by
measuring the length of migrated samples and calculate the Rf. Extrapolate the
log MW from the graph in (2) to find the samples molecular weight in kDa.

Sample Length migrated Rf Log MW Protein sample

(cm) (MW) in kDa

A 1.9 0.29 1.79 62

B 1.8 0.27 1.83 67

Chart Title
3

2.5

2 f(x) = − 1.86 x + 2.41

Log MV

1.5

0.5

0
0 0.1 0.2 0.3 0.4 0.5 0.6
Mobility(Rf)

1.9
1.88
1.88
f(x) = − 1.62 x + 2.26
1.86

1.84 1.83
1.82
Log MV

1.79
1.8
1.78
1.78

1.76

1.74

1.72
0.23 0.24 0.25 0.26 0.27 0.28 0.29 0.3 0.31
Mobility(Rf)
4. Finally, explain the similarity and the difference of DNA and protein gel Electrophoresis.
The DNA and protein gel Electrophoresis similar in the separation of molecules based on the
charge and size. The difference of them is DNA gel Electrophoresis staining with ethidium
bromide, while the protein gel Electrophoresis staining use Coomassie staining.

References:
https://ruo.mbl.co.jp/bio/e/support/method/sds-page.html
https://www.bio-rad.com/en-my/applications-technologies/protein-electrophoresis-methods?
ID=LUSOW4GRI