AGAROSE GEL

ELECTROPHORESIS
July 11, 2022
Objectives

At the end of the class, the students will be

able to:
• Explain the importance of agarose gel
electrophoresis.
• Perform the agar preparation for gel
electrophoresis.
• Explain the step-by-step procedure of
agarose gel electrophoresis
Introduction
• Agarose gel electrophoresis is a powerful separation method frequently used to
analyze DNA.
• The agarose gel consists of microscopic pores that can act as a molecular sieve.
• Samples of DNA can be loaded into wells made in the agarose gel and when an
electric field is applied, the DNA molecules are separated by the pores on the
gel according to their size and shape.
• After electrophoresis, the gel can be stained, and results can be analyzed
quantitatively by visualizing the gel with UV light in a gel imaging device. The
image is recorded with a computer operated camera and the intensity of the
band or spot of interest is measured and compared against standard or markers
loaded on the same gel.
Materials
• Isolated plasmid DNA from previous exercises
• 500ml Erlenmeyer flask
• Pipettor (P10, P100, P1000)
• Pipette tips
• Oven
• Gel Electrophoresis apparatus
• 1x TAE buffer
• UV transilluminator
• Agarose powder
• Hi-SYBr Safe Gel Stain
• Loading buffer
• Molecular weight marker
Agar Preparation
1. Prepare 1.5 % (w/v) agarose in 1X TAE.
2. After mixing the solution, place in the oven for
boiling.
3. Check the mixture. Swirl the flask for mixing.
You may choose to heat it again until all power
is dissolved.
4. Allow the agar to cool down. Then add the
DNA stain (Hi-SYBr Safe Gel Stain) before it
solidifies.
5. Transfer the agar to the mold before
solification.
6. Allow the gel to solidify within 15-20minutes.
AGAROSE GEL PREPARATION
Electrophoresis

7. Carefully remove the comb and mount the gel in

the electrophoresis tank.
8. Add enough buffer to cover the gel to a depth of
about 1 mm.
 Loading the sample to
the agarose gel
9. Mix the sample of DNA in the desired gel
loading buffer on the clean side of a parafilm.
10. Load the mixture into the well.
11. load an appropriate standard molecular
weight marker to serve as standard.
 Electrophoresis run
12. Run at optimum voltage ( =100 volts) until
the blue dye has run 3⁄4 of the gel.
13. View the gel under UV light to visualize the
DNA bands.
Electrophoresis Result