Aims and Objectives

Material and Methods

Materials: Onions, knives, chopping boards, gloves. Beakers, Blenders, Filters funnel and
filter paper. Ice baths, water bath at 600C. 15-20ml of ice cold ethanol per group, pasteur
pipette or glass rods, methylene blue. Coverslips/ slides

Procedure:

1. An onion was chopped into small pieces ( plastic gloves are worn to prevent
DNAases on the hands cutting the DNA in the material).
2. 50g of the chopped onion was transferred to a beaker.
3. 100 ml of Lysis Buffer* was added, and incubated for 15 minutes at 600C in a water
bath. This heat treatment softened the onion tissue and allowed penetration of the
lysis solution. It also denatured many enzymes that could interfere with the isolation
procedure.
4. The beaker was cooled by placing it in an ice bath for 6 minutes ( prevented
denaturation of the DNA).
5. The mixture was poured into a blender and homogenized for 45 seconds at low
speed, then 30 seconds at high speed.
6. The mixture was poured into a beaker and placed in an ice bath for 15 minutes.
7. The solution was passed though filter paper to obtain a clear filtrate liquid, which is
then poured into a beaker and placed in an ice bath for 15 minutes.
8. 15 – 20ml of ice cold ethanol was added immediately added to graduated cylinder by
SLOWLY pouring the ethanol down the side of the tube out of graduated CYLINDER.
A clear layer of ethanol was formed on top of the onion filtrate. DNA was not soluble
in ice cold ethanol. When ethanol was added to the mixture, all the components of
the mixture except for DNA stay in solution, while the DNA precipitated out.
9. The tube was let to sit for 3 – 5 minutes without disturbing it. Bubbles were formed
and DNA was precipitated out the solution. The DNA became visible as white strings
in the ethanol layer.
10. DNA was spooled by snagging it with a Pasteur pipette or glass rod
11. A small amount of DNA was put on slide. A drop of methylene blue was added. A
cover slip was put. Observations and drawings have been made.