2/17/2025
EXTRACTION OF DNA FROM BANANA
The experiment has 2 parts
(i) Isolation of Genomic DNA from banana
(ii) Observation of Electrophoresis apparatus and stained
prokaryotic and eukaryotic DNA Gel (as given in later
image )
Nucleic Acids
• Nucleic acid is an important class of macromolecules
found in all cells.
• Major nucleic acids are –
• DNA – Deoxy-ribonucleic acid (in Nucleus, Chloroplast &
Mitochondria) and
• RNA – Ribonucleic Acid (along DNA and in cytoplasm)
• Function - storage and transfer of hereditary information
• It is a polymer that consists of Nucleotide monomers
• Nucleotides consists of : Nucleosides (Nitrogenous base +
5 – Carbon sugar) + One Phosphate group
• Elemental composition: Carbon, Hydrogen, Oxygen,
Nitrogen and Phosphorous
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Experiment 1: PRINCIPLE of DNA Extraction
• DNA extraction involves lysing the cells (breaking the cell and nucleus membrane) and
solubilizing DNA, which is followed by chemical or enzymatic methods to remove
macromolecules, lipids, RNA, or proteins.
• Banana pulp: Many cells – large amount of DNA
• NaCl + Cedopol (salt + liquid detergent) – Disrupts the cell membrane’s phospholipid bilayer
by reacting with the phosphate group of the phospholipid.
• Cellular components are released into the buffer.
• Now, Na+ will bind to the phosphate molecules in the DNA and shield them.
• DNA is loosely bound together because of the shielding.
• The alcohol helps to precipitate the DNA from the solution.
• Buffer solution is denser than alcohol, the alcohol will float on top.
• Boundary between the two is called Interface.
• The DNA precipitates at the interphase
• Small fragments of DNA and degraded RNA usually contaminate the chromosomal DNA
during extraction procedures
• RNA are also precipitated by the alcohol, but have little tendency to spool on the
loop because they are too short and form finer, more uniform precipitates
• Spooling can be viewed as a method that partially purifies and concentrates high
molecular weight DNA
• Precipitate can be collected and re-dissolved in a smaller volume, to concentrate
nucleic acids
• Alcohol precipitations also remove small molecules, such as buffer salts, sugars and
amino acids from nucleic acid precipitations since they remain in solution
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Materials Required
Sodium Chloride Banana
Cedopol Muslin cloth
Distilled water Mortar and Pestle
100 ml and 250 ml Beaker Tooth pick/ Glass rod
Ice cold isopropanol Eppendorf (1.5 ml)
Procedure
1. Detergent Preparation:
Mix the following contents in a clean 100 ml beaker
a. 80 ml distilled water
b. 3g sodium chloride
c. 10 ml of Cedopol and make up the volume to 100 ml
Each group will be given 50 ml of Detergent.
(Will already be prepared by Naresh ji & will be placed on the working bench of each group)
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Procedure
2. Preparation of the Lysate from Banana: To be done by the students
Mash 50g of banana in a mortar and pestle until a smooth pulp is obtained.
Transfer the pulp in a 250 ml beaker and add 50 ml of detergent solution to it gradually while
mixing. Mix it gently but thoroughly.
Place the beaker containing the homogenized banana in a 60°C water bath for 15 minutes and
remove the beaker to an ice-water bath for 5 minutes to cool the pulp.
Place a muslin cloth on the mouth of the beaker and secure it with a rubber band.
Carefully pour the homogenized banana solution into the muslin cloth. After 5-10 mins, you will
observe ~10 ml of filtered solution.
Procedure
3.Precipitating the DNA and Spooling: 30ml Chilled Isopropanol will be provided to each
group by instructor after the filtration step (Kept in fridge)
Add 30 ml of ice cold isopropanol in the beaker and gently pipette out the 10 ml filtered solution
down the side of the beaker. Since the DNA/buffer solution is denser than the alcohol, the alcohol
will float on top. The boundary between the two is called an interface. DO NOT MIX IT.
Insert the tooth pick/ glass rod into the beaker. Carefully swirl the tooth pick/ rod and wind (spool)
the DNA that comes out of solution on to the loop. These are not single DNA molecules, but
thousands of molecules. After a minute of spooling, slowly remove the tooth pick/ rod from the
tube. A clear, viscous, clotted mass of DNA will adhere to the rod.
Observe the spooled DNA. Draw a picture of both interface formed, DNA in beaker as well as
spooled DNA.
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DNA Results
Experiment Part B: Electrophoresis
• Technique for separation or resolving
charged molecules in a mixture under the
influence of applied electric field
• Charged molecules migrate in the electric
field at a speed determined by their charge
to mass ratio
• Movement of molecules is due to the
voltage applied across the electrodes, leads
to current flow across gel
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Agarose Gel Electrophoresis
• Gel serves as porous matric and behaves as a molecular sieve.
• Agarose, made of agarobiose, is a natural colloid extracted from sea weed
• Pore size is large, hence used to separate nucleic acids
• Electrophoresis buffer: Allow current to flow, while resisting any pH changes that
may happen
• Running buffer for nucleic acids is TAE/ TBE buffer
• Loading dye (with sample for tracking it) eg: Bromophenol blue
• EtBr: chelating agent, under UV for band visualization (added to buffer)
• Polyacrylamide (polymer of acrylamide and bis-acrylamide) can also be used for
DNA and usually for Protein
Agarose Gel Image and its Explanation
Lane 1 – Genomic DNA (Lambda 1 2 3 4
Phage DNA).
Lane 2 – Genomic DNA (Lambda
Phage DNA) restriction digested with
Hind 3 enzyme
Lane 3– Genomic DNA (Lambda
Phage DNA) restriction digested with
Hind 3 and EcoRI enzyme
Lane 4– Plasmid (pRSETA) DNA
(In Low Concentration)
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Agarose Gel Image and its Explanation
Relaxed Circular (Nicked) – Plasmid with one DNA strand cut or nicked.
Nick or cut in 1 DNA strand, leaves a large,
floppy circle, causing this structure to take up
the most volume, thereby migrating most
slowly through the gel.
Supercoiled Form - Comparatively, supercoiled forms migrate the fastest
because of their conformation (small and compact) as
they sustain less friction than others.
Linear DNA - DNA helix is cut in both strands at the same place.
Linear DNA runs second to supercoiled DNA because Different Conformations of
Plasmid DNA as seen on
of its less compact form compared to supercoiled DNA.
Agarose gel
Genomic DNA - Since it is of Very High molecular weight (in Mb), therefore, its size is not comparable
to the marker.
