Isolation of Genomic DNA
Aim:
Isolation of genomic DNA from human cheek cells
Principle:
DNA is present in every living cell, it can be isolated from hair roots, cuticle of nails, skin and
even cheek cells can be lysed to release nucleus. SDS /detergent help in dissolving liquid from
cell membrane which causes cell lysis and over release of organelles and their inclusions are
insoluble in the saline solution, so they settle in the beaker when chilled ethanol is poured.
Solubility of DNA decreases which causes precipitation in the form of spool. This precipitate is
lighter than solution and can be seen floating.
Materials required:
Normal saline solution (0.9% NaCl) (non-ionized), 95% ethanol (ice cooled), glass rod, beaker,
detergent (SDS), buffer (5% NaCl), papaya or Pineapple juice.
Procedure:
1. Collect the cheek cells in test tube by chewing them in saline solution and add detergent (2-3
ml) and add 3 to 4 ml of papaya juice.
2. Shake the solution and keep it undisturbed for 7 to 10 minutes.
3. Pour chilled ethanol in solution by the wall of the test tube and rub the test tube gently.
4. Collect the DNA spool by glass rod in the 5% NaCl buffer.
5. Add 1 to 2 drops of phenol red indicator.
Yellow colour shows the presence of DNA
Pink colour shows the absence of DNA.
Result:
DNA is precipitated in the form of spool and this is collected in 5% NaCl buffer.
Precautions:
1. Use chilled ethanol only.
2. Wear lab-coat while performing the experiment.
3. Chilled ethanol should be poured along the walls of the test tube.
Paper Chromatography
Aim:
To demonstrate the principle of paper chromatography.
Principle:
Chromatography is a process through which various substances in a mixture are separated from
each other and identified. Separation of mixture i.e. dyes is based on liquid /liquid partitioning
of dyes. The partitioning takes place between water molecules (adsorbed) (stationary phase) to
cellulose matter of the paper and organic (mobile) phase.
The rate of movement of molecules is known as Rf value (Retention factor) and is expressed and
calculated by the following formula:
The Rf value for a substance is similar under particular set of conditions and therefore, Rf value
is crucial in identifying and separating a substance for potential materials of different sources.
Requirements:
Petridish with cover, capillary tube, beaker, Whatman paper No. 1, Acid fuchsin, Aniline blue
orange-G, n-butanol, Distilled water
Procedure:
1. Take a circular Whatman paper with the diameters slightly larger than the petridish.
2. Take the test material with the help of a capillary tube and place a very small drop of it on the
centre of the paper, Allow the drop to dry completely.
3. Take a thick thread and pass one end of it through the centre of the paper and let the other end
touch the bottom of the dish when the paper is placed on it.
4. Fill the dish with n-butanol up to approximately one quarter mark.
5. Place the paper with the spot on top of the dash and let the thread lie dipped in the solvent.
6. The disc should completely cover the petridish.
7. Cover the apparatus and wait for about 2 to 3 hours.
8. Remove the paper and mark the distance travelled by the mobile phase from the centre of the
paper. Dry the paper.
9. Examine the circular bands of individual dyes which separate and travel to their specific
positions on the stationary phase. Mark the distance travelled by the dyes from the spot.
Result:
The individual bands of each dye can be seen and identified by circular paper chromatography.
Rf value of Acid fuschin =
Rf value of Aniline blue =
Rf value of Orange G =
