Bio lab 2021

LAB 3 – AGAROSE GEL ELECTROPHORESIS

BITS Pilani
Pilani Campus

Department of Biological Sciences, BITS Pilani, Pilani Campus

 What is Gel electrophoresis?

• Gel electrophoresis is a technique commonly used

in laboratories to separate charged molecules
like DNA, RNA and proteins according to their
size or charge.

• Charged molecules move through a gel when an

electric current is passed across it.

• DNA is negatively charged, therefore, when an

electric current is applied to the gel, DNA will
migrate towards the positively charged electrode.

Department of Biological Sciences, BITS Pilani, Pilani Campus

 Principle

• An electric current is applied across the gel so that one end of the gel has a
positive charge and the other end has a negative charge.

• The gel consists of a permeable matrix, a bit like a sieve, through which
molecules can travel when an electric current is passed across it.

• Smaller molecules migrate through the gel more quickly and therefore travel
further than larger fragments that migrate more slowly and therefore will travel
a shorter distance. As a result the molecules are separated by size.

Department of Biological Sciences, BITS Pilani, Pilani Campus

Agarose gel electrophoresis

https://www.jove.com/t/3923/agarose-gel-electrophoresis-for-the-separation-of-dna-fragments

Department of Biological Sciences, BITS Pilani, Pilani Campus

 Preparation of Agarose gel

• Prepare 1 % agarose gel (weigh 0.4 gram and add in 40 ml of 1X TAE buffer)

• Boil/ microwave in oven for 1-3 mins until agarose get dissolved completely.

• Allow agarose solution to cool down(when you can comfortably keep your hand on the
flask) and Add 2 µl of EtBr in 40 ml gel preparation. Be careful with the EtBr fumes.

• Pour the agarose into a gel tray with the well comb in place. Allow it to solidify.

Concentration of agarose- agarose gel matrix acts like a sieve. % of agarose determines
size of the sieve matrix. The greater the percentage of agarose, the smaller the linear DNA
that can be resolved. Larger molecules can be resolved with lower concentration of agarose

Department of Biological Sciences, BITS Pilani, Pilani Campus

 Role of TAE buffer and Ethidium bromide dye

• TAE buffer is a buffer solution containing • Ethidium bromide is a molecule commonly used to
a mixture of Tris base, acetic acid and visualize DNA in agarose gel electrophoresis
EDTA. pH 8.3 (mention importance of pH ) experiments.
• Tris and Acetic acid- maintaining the pH • It binds to DNA and fluoresces under the proper
and buffering capacity. conditions (UV light) and known to be an
• EDTA- chelates the divalent cations intercalating agent.
inhibiting the Dnase activity
• The flat structure of ethidium bromide allows it to
• It is used as both a running buffer and in
intercalate, or insert, between nitrogenous bases of
preparation of agarose gel.
a DNA molecule.
• Buffer provides ions for conductivity and
maintains pH so that DNA remains stable. • Ethidium bromide is that it is a potent mutagen.
Avoids heating and melting of gel. Solutions must be handled with extreme caution
and decontaminated prior to disposal

Department of Biological Sciences, BITS Pilani, Pilani Campus

Role of Ethidium bromide dye

Department of Biological Sciences, BITS Pilani, Pilani Campus

Loading samples and Running on Agarose Gel

Once solidified, place the agarose gel into the electrophoresis unit
filled with TAE Buffer.
Add loading dye to each of your DNA samples.
Carefully load the DNA ladder into the first well and samples into
the additional wells of the gel (prevent bubbles or buffer from
entering the tip)
Run the gel at 80-100 V until the dye line is approximately 75-
80% of the way down the gel.
A typical run time is about 0.5-1.5 hours, depending on the gel
concentration and voltage.

Department of Biological Sciences, BITS Pilani, Pilani Campus

 Role of loading dye and DNA Ladder

• Loading dye is added to DNA sample to give • DNA ladder, a molecular-weight size marker,
it color to the naked eyes. is a set of standards that are used to identify
• Helps with gel loading and allows you to the approximate size of a molecule run on a
gauge how far the DNA has migrated; gel during electrophoresis.
• 6X loading dye contains: • It contains DNA fragments of known length,
1. 0.25% (w/v) Bromophenol blue- Tracking making them suitable for estimating the
dye to monitor progress of gel fragment length of concurrently run samples.
2. 0.25% (w/v) Xylene cyanol- tracking dye • Available as 50bp, 100bp, 1000bp(1Kb) or
used in combination with bromophenol blue 3000bp
to increase its effectiveness and quality of
result.
3. 30% Glycerol- increases the density of the
DNA sample so that it can settle to the
bottom of the well, instead of diffusing in
the buffer.

Department of Biological Sciences, BITS Pilani, Pilani Campus

https://www.jove.com/v/5057/dna-gel-electrophoresis

Department of Biological Sciences, BITS Pilani, Pilani Campus

 Analysing the Gel
 Using the DNA ladder in the first lane as a guide you can infer the size of the DNA in your
sample wells.

EtBr stained gel

Department of Biological Sciences, BITS Pilani, Pilani Campus
Activity : part 1

Video : Agarose gel electrophoresis

Department of Biological Sciences, BITS Pilani, Pilani Campus

 Applications of gel electrophoresis

• In the separation of DNA fragments for DNA fingerprinting to investigate crime

scenes.

• To analyze results of polymerase chain reaction.

• To analyze genes associated with a particular illness.

• In DNA profiling for taxonomy studies to distinguish different species.

Department of Biological Sciences, BITS Pilani, Pilani Campus

THANK YOU

Department of Biological Sciences, BITS Pilani, Pilani Campus