DNA Extraction 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 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 allow 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 the lysing or
homogenization buffer.
This procedure is used to extract large amounts of DNA from onions, and similar
protocols are used to isolate DNA from other sources, such as a blood sample.
In addition to the DNA extraction from onion, you will also be performing a DNA
extraction from Arabidopsis, using a commercially available kit. We will discuss the
pro’s and con’s of each method.
Materials and Equipment
Blender
50% ethanol
Ice bucket
50 ml conical tube
250 ml beaker
60°C water bath
Chopped onions
Homogenization Buffer
15 ml conical tube
Glass rod
Isopropanol
Cheesecloth
Funnel
500 ml beaker
Procedure
1. Place 10 ml chopped onions in 50 ml conical tube.
2. Add 25 ml homogenization buffer to the onions. Mix well by inverting the tube several
times.
3. Incubate mixture for 10 minutes at 60°C.
4. Cool mixture by swirling tube in an ice bath. Swirl tube until it feels cool to touch.
5. Combine your mixtures with that of two other groups, into a blender and fasten the lid.
Homogenize for 30 seconds at low speed. Mixture will be very foamy.
6. Pour mixture into 500 ml. Swirl beaker gently in ice bath to cool the mixture. Keep the
beaker in ice, such as in an ice chest or bucket, until it feels cold to touch. There should
be liquid forming beneath the foam in the beaker.
7. Pour mixture through folded layers of cheesecloth in a funnel into 250 ml beaker.
8. Divide the filtrate into clean 50 ml conical tubes. Each group should receive a tube,
and each tube should contain 10-15 ml onion liquid.
9. Add equal volume of isopropanol to onion mixture. Cap the tube and mix gently by
rocking back and forth until the DNA precitipates.
10. Spool out the stringy DNA by rotating a glass rod in one direction to wrap the DNA
around the stick.
11. Gently ease the DNA into a tube with 50% ethanol.
Questions
1. What is the purpose of the homogenization buffer? Describe the role to the different
chemicals.
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?
Homogenization Buffer Recipe
50.0 g Sodium lauryl sulfate (SDS)
8.8 g Sodium chloride
4.4 g Sodium citrate
0.3 g Ethylenediamine tetraacetic acid (EDTA)