Aim: AGAROSE GEL ELECTROPHORESIS

(Here we are going to determine the size and quality of the extracted DNA from
Blood.)

Introduction and principle of the experiment:

Electrophoresis is a technique used to separate ad sometimes purify
macromolecules especially proteins and nucleic acids that differ in size, charge
or conformation. As such, it is one of the most widely used technique in
biochemistry and molecular biology. When charged molecules are placed in an
electric field, they migrate toward either the positive or negative pole according
to their charge. In contrast to proteins which can have either a net positive
charge, nucleic acids have a consistent negative charge imparted by their
phosphate backbone and migrate toward the anode.
The most commonly technique for nucleic acid separation is agarose gel
electrophoresis. Agarose gels are ideal for RNA and DNA because agarose is
inserted to these molecules and the molecules are easily recovered from the gel
for further use. Nucleic acids will separate by size in agarose without the need
for chemical modification. Agarose is easily prepared by dissolving agarose
(refine from sugar) in heated buffer solution.

Materials:
 Agarose powder
 10xTAE/TBE buffer
 Ethium Bromide
 Loading dye
 100 ml Erlenmeyer flask
 1kb DNA ladder
 Deionised distilled water
 Gel chamber with gel tray and comb.

Procedure:
  Prepared 0.8% agarose gel by weighing out of the agarose powder and
transfer into 100mL Erlenmeyer Flask.
 Added 50mL of 10x TAE/TBE buffer and swirl the flask to ensure
homogenous distribution of the agarose powder in the buffer.
 The mixture is heated until it begins to boil. Swirl the flask very well to
make sure all agarose is dissolved in the buffer (no solid powder form
should be visible).
 Continue until a clear translucent solution is formed.
 Set aside to cool down just enough to where the solution will not melt the
gel tray.
 While the gel solution is cooling, filled the gel chamber with
10xTAE/TBE.
 Once the agarose buffer has cooled slightly, added 2µL of ethidium
bromide into the solution and poured into the gel tray with well comb
fixed properly.
 After the gel has cooled completely and solidified the combs can be
removed and the tray inserted properly into the gel chamber.
 To cover the gel, poured enough 10xTAE/TBE buffer into the chamber
and filled the wells.
 Into the wells, pipette 6µL of 1Kb DNA ladder(mixed with Loading dye)
and 12µL of samples (mixed with Loading dye)
 Closed the lid of gel chamber.
 For 30 minutes, run the gel at 100V.
 Under the UV light, visualized the gel.

Critical thinking

 What is gel electrophoresis and why is it used?

Gel electrophoresis is a laboratory method used to separate mixtures of DNA,
RNA, or proteins according to molecular size. In gel electrophoresis, the
molecules to be separated are pushed by an electrical field through a gel that
contains small pores. In other words gel electrophoresis is a technique used to
separate DNA fragments (or other macromolecules, such as RNA and proteins)
based on their size and charge. Using electrophoresis, we can see how many
different DNA fragments are present in a sample and how large they are relative
to one another.

 What gel is used in gel electrophoresis?

The types of gel most typically used are agarose and polyacrylamide gels. Each

type of gel is well-suited to different types and sizes of the
analyte. Polyacrylamide gels are usually used for proteins and have very high
resolving power for small fragments of DNA (5-500 bp).

 What are the advantages of gel electrophoresis?

The advantages are that the gel is easily poured, does not denature the samples.
The samples can also be recovered. The disadvantages are that gels can melt
during electrophoresis, the buffer can become exhausted, and different forms of
genetic material may run in unpredictable forms.