DNA Extraction from Onion - Student Handout "Chemical version"

Isolating DNA from cells is an important first step in doing DNA analysis. 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. You will learn how familiar chemicals are used to purify DNA.

Materials and Equipment

Per class

Blender

(60 to 70°C) water bath

Isopropanol

50% ethanol

Chopped onions

Onion lysis solution

Cheesecloth

Ice chest containing ice

Per group

Ice bucket or beakers with ice

Funnel

50 ml conical tube

15 ml conical tube containing 50% ethanol

500 ml beaker

250 ml beaker

Plastic sticks

Procedure

1. Measure about 10 ml chopped onions in 50 ml conical tube. Add 25 ml onion lysis solution to tube. Mix well by shaking tube several times.
   
   Onion lysis solution contains: sodium lauryl sulfate, sodium choloride, sodium citrate, and ethylenediamine tetaacetic acid (EDTA).
   
   
2. Incubate mixture for 10 minutes at 60°C.
   
   
3. Cool mixture by swirling tube in an ice bath. Swirl tube until it feels cool to touch.
   
   
4. Combine up to four cooled mixtures into a blender and fasten the lid. Homogenize for 30 seconds at low speed. Mixture will be very foamy.
   
   
5. Pour mixture into 500 ml or 1000 ml beaker. 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.
   
   
6. Pour mixture through folded layers of cheesecloth in a funnel into 250 ml beaker. Save the onion liquid in the beaker, taking care to leave the foam behind in the cheesecloth. If the class period is almost over, the onion liquid in the beaker can be covered and stored in the refrigerator overnight.
   
   
7. Divide the onion liquid into clean 50 ml conical tubes. Each group should receive a tube, and each tube should contain 10-15 ml onion liquid.
   
   
8. Add equal volume of isopropanol to onion mixture. Be sure the tube is capped. Mix tube gently by rocking back and forth until a white, stringy mass appears. This mass is the onion DNA.

9. Spool out the stringy DNA by rotating a plastic stick in one direction to wrap the DNA around the stick. Gently ease teh DNA into a tube with 50% ethanol.

Questions

1. Why is detergent used? Why is heat used with the detergent?
   
   
2. Why is the onion mixture processed through the blender?
   
   
3. Why is isopropanol added to the onion liquid?
   
   
4. What does the purified DNA look like?
   
   
   
   

BIOTECH Project Department of Molecular and Cellular Biology The University of Arizona August 3, 1998 Designed by: Ken Kubo, Ph.D. Nadja Anderson, Ph.D. nadja@email.arizona.edu http://biotech.biology.arizona.edu