DNA Isolation Lab
Purpose: To provide you with the opportunity to isolate and observe DNA. You will
gain an understanding of how a fractionation procedure is carried out, the roles of each
substance involved, and how easy it actually is to isolate DNA.
Background: The process of isolating DNA from a cell is the first step for many
laboratory procedures in biotechnology. One must be able to separate the DNA from the
unwanted substances of the cell gently enough so that the DNA is not broken up or
shredded.
A filtrate is made of onions treated with salt, distilled water and detergent (SDS). An
onion is used because it has a low starch content which allows the DNA to be seen more
clearly. The salt shields the negative phosphate end of DNA which allows these ends to
come closer so they can precipitate out of a cold alcohol solution. The detergent causes
the cell membrane to breakdown by emulsifying the lipids and proteins of the cell and
disrupting the polar interactions that hold the cell membrane together. The detergent then
forms complexes with these lipids and proteins, causing them to precipitate out of
solution. Collectively, the salt solution and detergent are referred to as a lysing or
homogenization buffer.
Materials and Equipment:
Blender
Ice bucket
Cheesecloth
Funnel
50ml conical tubes
500ml beaker
250ml beaker
15ml conical tubes
Homogenization buffer
Chopped onion
Isopropanol
50% Ethanol
Procedure:
1. Place ~10ml chopped onions into a 50ml conical tube.
2. Add 25ml homogenization buffer to onions in the 50ml tube. Mix well by inverting the
tube several times.
3. Incubate mixture for 10 minutes at 60oC.
4. Cool mixture by swirling tube in an ice bath. Swirl tube until it feels cool to touch.
5. Combine your mixture with that of 2 other groups in a blender and fasten the lid.
Homogenize for 30 seconds at low speed. Mixture will be very foamy.
6. Pour mixture into a 500ml beaker. Swirl beaker gently in an ice bath to cool the
mixture. Keep the beaker on ice until it feels cool to the touch.
7. Filter mixture through several folded layers of cheesecloth in a funnel into a 250ml
beaker.
8. Divide the filtrate into clean 50ml conical tubes. Each group should receive a tube, and
each tube should contain 10-15ml of filtrate.
9. Add an equal volume of isopropanol to filtrate. Cap tube and mix tube gently by
rocking tube back and forth until the DNA precipitates.
10. Spool out the DNA by rotating a glass rod in one direction.
11. Gently ease the DNA into a 15ml tube with distilled water.
Questions:
1. What is the purpose of the lysing/homogenization buffer? Describe the role of the
different chemicals if possible.
2. Describe the appearance of the DNA precipitate from step 9.
3. The fact that you can wind the DNA onto a glass rod implies what about its physical
nature?
4. Was all that stirring necessary? Would the experiment have worked as well without the
10 minutes of stirring with soap solution? Why or why not?