Agarose Gel Electrophoresis PDF

Aim: To resolve DNA fragments on the basis of their molecular weight and charge
Introduction:
In molecular biology, agarose gel electrophoresis is a commonly used method for sorting and
analysing DNA molecules according to their size and charge. It is an effective and adaptable
instrument that has a wide range of uses, from fundamental research to clinical diagnosis.
A step-by-step tutorial for the preparation of agarose gels, loading of DNA samples, running of
the electrophoresis, and viewing of DNA bands are provided in the practical manual for agarose
gel electrophoresis. It covers the basic concepts of agarose gel electrophoresis as well as
crucial factors for quality assurance and problem-solving.
The manual contains comprehensive instructions on how to prepare agarose gels with various
concentrations, load DNA samples, and run electrophoresis under various circumstances.
Additionally, it discusses the staining and viewing of DNA bands using different DNA-specific
dyes, including ethidium bromide and SYBR Green.
The booklet also offers instructions for proper handling and disposing of dangerous substances
like ethidium bromide and the electrophoresis buffer. In order to guarantee the accuracy and
reproducibility of results, it also emphasises the significance of quality control methods, such as
utilising the proper DNA size markers and doing control samples.
In general, the practical manual for agarose gel electrophoresis is a vital tool for academics, lab
personnel, and researchers that utilise this method for DNA analysis. It offers a thorough
explanation of agarose gel electrophoresis theory, practice, and safety issues, making it
possible to analyse DNA molecules with confidence and accuracy.
Principle:
The foundation of this method is the response of DNA molecules to an electric field, which
causes them to move across a gel matrix. When heated and chilled, the polysaccharide agarose
from seaweed creates a matrix that resembles gel. It is possible to separate DNA molecules of
various sizes by altering the concentration of agarose used in the gel formation process.
Smaller pores produced by higher agarose concentrations enable greater separation of smaller
DNA fragments, whereas bigger pores produced by lower agarose concentrations enable larger
DNA fragments to flow through the gel.
The negatively charged DNA molecules migrate towards the positively charged electrode at the
other end of the gel when an electric current is given to wells containing the DNA sample at one
end of the gel during the electrophoresis procedure. The DNA fragments are separated based
on size as the DNA molecules move through the gel matrix, with smaller pieces migrating
quicker and moving farther down the gel than bigger fragments.
Utilising DNA-specific dyes, such as ethidium bromide, which intercalates between the DNA
base pairs and fluoresces under UV light, it is possible to see the separated DNA after
electrophoresis is finished. The size of the DNA fragments in the sample can be determined by
comparing the resultant DNA bands to DNA size markers of known size.
In general, agarose gel electrophoresis is an effective and flexible method for the separation
and examination of DNA molecules. To get precise and repeatable results, it's crucial to adhere
to the protocol and quality control procedures outlined in the practical manual.
Materials:
Reagents and solutions:
● 1X TAE Buffer
● Agarose
● Ethidium Bromide
● DNA Loading Dye
● DNA Sample
Equipment and Glassware: Casting Tray, Comb, Flask, Cello tape, Paraffin paper, Micropipette,
Electrophoresis tank, Power pack,UV Box.
Procedure:
● Preparation of Agarose Gel
1. Prepare 1X TAE buffer.

2. Add 0.2 gm of ultra-pure agarose to 20 ml of 1X TAE buffer in a flask.
3. Swirl the flask to mix.
4. Microwave solution for 30 seconds to 1 min. Remove the flask with gloves and
swirl.
5. Allow the solution to cool for 1 min.
6. Add 1 μL of ethidium bromide to the solution and swirl.
7. Pour the solution into the Casting tray.
8. Insert the comb into the top of the gel and allow the gel to solidify for 30 min.
● Running Gel Electrophoresis
1. Once the gel has solidified, carefully remove the comb by pulling it straight up
2. Insert the tray into the electrophoresis chamber/tank containing 1X TAE Buffer.
3. Ensure the gel is in the correct orientation, with the negative/black electrode
above the wells, so the DNA runs toward the positive/red electrode.
4. On a paraffin paper, place a few drops of DNA loading dye.
5. Using a micropipette take an appropriate amount of sample DNA and mix well
with the loading dye using pipette in and pipette out. Avoid bubbles.
6. Load samples into wells.
7. Place the lid on the tank and turn on the power supply at 100 Volts.
8. After 1-1.5 hrs, turn off the power supply off, disconnect the electrodes from the
power source, and then carefully remove the tray containing the DNA sample..
9. Visualize the gel under UV light.
Result: DNA bands were visible under UV light after Agarose Gel Electrophoresis

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Aim: To resolve DNA fragments on the basis of their molecular weight and charge

Reagents and solutions:

● Preparation of Agarose Gel

1. Prepare 1X TAE buffer.

● Running Gel Electrophoresis

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