unisel

UNIVERSITI SELANGOR

FACULTY OF SCIENCE AND
BIOTECHNOLOGY
(FasBIO)
MOLECULAR TECHNIQUES BBS 1231
Practical 5: Agarose gel electrophoresis
(LECTURER: MDM. Yasotha a/p Sundaraj)
GROUP 3
Devaraj a/l Ravindran 4111018161
Jeevitha a/p Tana sakaran 4111016861
Sujatha a/p kanniappan 4111009531
Premkumar a/l Subramaniam 4111017391
Theevindran a/l Kesavan 4111017311
Paveanthen a/l Ramachandran 4111023071
Mohamed Mustafa Osman 4102008031


INTRODUCTION:
Gel electrophoresis is using a gel as a sieving medium during electrophoresis. Gel electrophoresis is most
commonly used for separation of biological macromolecules such as deoxyribonucleic
acid (DNA), ribonucleic acid (RNA) and protein. Gel electrophoresis can be used for separation of
nanoparticles. Electrophoresis refers to the movement of a charged particle in an electrical field. Gels
suppress the thermal convection caused by application of the electric field, and can also act as a sieving
medium, retarding the passage of molecules. Gels can also simply serve to maintain the finished
separation, so that a post electrophoresis stain can be applied. DNA Gel electrophoresis is usually
performed for analytical purposes, often after amplification of DNA through PCR.
Gel refers to the matrix used to contain, then separate the target molecules. In most cases, the gel is
a cross linked polymer whose composition and porosity is chosen based on the specific weight and
composition of the target to be analyzed. When separating proteins and small the gel is usually composed
of different concentrations of acrylamide and a cross-linker, producing different sized mesh networks
of polyacrylamide. When separating larger nucleic acids the preferred matrix is purified agarose. In both
cases, the gel forms a solid, yet porous matrix. Acrylamide, in contrast to polyacrylamide, is
a neurotoxin and must be handled using appropriate safety precautions to avoid poisoning. Agarose is
composed of long unbranched chains of uncharged carbohydrate without cross links resulting in a gel
with large pores allowing for the separation of macromolecules and macromolecular complexes.
Electrophoresis refers to the electromotive force (EMF) that is used to move the molecules through the
gel matrix. By placing the molecules in wells in the gel and applying an electric field, the molecules will
move through the matrix at different rates, determined largely by their mass when the charge to mass ratio
of all species is uniform, toward the anode if negatively charged or toward the cathode if positively
charged.

AIM:
To conduct agarose gel electrophoresis
MATERIALS:
TBE Buffer (10)
108g Tris Base
55g boric acid
44ml 0.5M EDTA solution
1L ddH
2
O
Adjust the final pH to 8.0
( the stock solution was diluted to 1 as working solution)

6 Loading dye (Fermentas/Promega)

Agarose powder

Standard DNA Ladder marker

Red Safe solution (staining agent)
METHOD:
1. 0.3g of agarose powder was weighed and added into 30ml of 1 TBE buffer in a small
Erlenmeyer flask.
2. The flask with agarose was micro waved and checked occasionally to see whether all the
agarose has melted. The solution was not allowed to boil.
3. The flask was cooled by running cold tap water along the outside of the flask then 3 micro liter
of Red safe solution was added.
4. the agarose was gently poured into the mold when it is warm to touch. (The mold was prepared
prior to the pouring of agarose. The comb was placed before pouring the agarose and care was
taken to prevent bubble formation during the process.)
5. The gel was left to solidify.
6. The gel was ready to use, once it solidified.
7. The comb was gently removed by pulling it evenly upward.
8. The gel mold placed inside the electrophoresis chamber and the gel was covered with running
1TBE.
9. DNA samples were mixed with loading dye on a parafilm. The sample was diluted by mixing 5
micro liter of the sample with 1 micro liter of 6x loading dye.
10. The sample mixture and the DNA ladder were carefully loaded into respectively wells by using
pipette.
11. The cover to the electrode box was placed and the wire was plug to the power pack. The gel
runs at 120V for 45minutes to visualize the DNA.
12. The power pack was switched off once the loading dye has reached approximately of the gel.
13. The gel was carefully removed from the mold and placed it into the staining box containing Red
safe solution.
14. The gel was stained for approximately few minutes (not more than 5 minutes).
15. The stained gel was placed on a transilluminator. The UV light was switched on and the DNA
band was visualized. A photograph of gel with the DNA bands was snapped.
16. Once documentation of the gel has been carried out, the gel was disposed in a proper waste
bucket. Finally, the surface of the transilluminator was cleansed with distilled water.




OBSERVATION:

The first band is the standard DNA ladder maker, used as a reference to estimate the sizes of the
unknown DNA molecules. The last band is not displayed clearly because the sample was contaminated
with substance like protein.
DISCUSSION

1. TBE buffer (Tris-Borate-Edta) provides an ionic solution that allows current to pass through the
water. The Tris part is useful to keep the DNA deprotonated in solution. EDTA protects nucleic
acids from degradation. It removes certain metal ions from enzymes that would destroy the
nucleic acid, thus inactivating the enzymes.

2. Difference between TBE and TAE buffer:
The ion strength of TBE is higher than TAE buffer, thus solutes in TBE move faster in TAE.
Moreover, temperature of gel increases using TAE-gel for a long time, other than that, the pH
might significantly decrease because of the Tris base. Fluorescent moiety of labeled DNA might
go bad in the conditions of high temperature and lowered pH. Buffering capacity of TBE is also
greater than that of TAE.

3. Loading dye:
The loading dye helps to add on weight to the DNA, thus the DNA can sink into the bottom of
the wells and not float in the buffer solution. It also acts as an indicator which shows when to
switch of the power supplied to the electrophoresis chamber. Then, it also makes the DNA
visible to the naked eye, giving it a purplish colour and I can also be seen more clearly on a
transilluminator.

4. Standard DNA ladder maker:
It is a solution of DNA molecules of various lengths. It is applied to an agarose gel as a
reference to estimate the size of unknown DNA molecules.

5. Red safe solution:
It is a kind of reagent which is commonly used as a fluorescent tag in molecular techniques
especially in agarose gel electrophoresis. When it is exposed to ultraviolet light, it will fluoresce
with orange-purple colour, its intensity will increase after binding to DNA.
6. Agarose powder:
The main function of agarose is to serve as a matrix which resolves different sizes of DNA
fragments. Higher concentration of agarose facilitates separation of small DNAs, while low
agarose concentration allow resolution of larger DNAs.

CONCLUSION:
The gel electrophoresis method was carried out successfully.
REFERENCE:
1. Title: Comparison between TBE and TAE buffer. Accessed from: http://biowiki.edu-
wiki.org/en/comparison_between_tae_and_tbe. Accessed date: 30/9/2011.
2. Title: function of loading dye. Accessed from:
http://wiki.answers.com/Q/What_are_the_functions_of_the_loading_dye_in_electrophoresis.
Accessed date: 30/9/2011.