Biological Principles Lab: Strawberry DNA Extraction PURPOSE

Name: ______________________________________ Section: _____________________________________

To isolate genomic DNA from the eukaryote Fragaria (the strawberry). To observe the effect of alcohol on DNA molecules.

INTRODUCTION This lab activity will make use of a common procedure for the extraction of DNA from living cells via alcohol precipitation. While commercially-available kits are used widely in biomedical researchers, the underlying concept is straightforward and can be easily performed with minimal equipment. A mild detergent is added is to the cell suspension or tissue sample. The detergent solubilizes the plasma membranes of the cells and produces a crude cell lysate. Depending on the purity of the sample required, an enzyme called a protease is then added to the lysate, which digests any proteins in the sample (most notably the histones that are complexed with the genomic DNA). Ice-cold alcohol is then added which precipitates both the DNA and any salts that are ionically-bonded to it. The salts can be eliminated by additional alcohol washes, if necessary. Todays procedure will not result in a particularly pure DNA extraction, but both the DNA (and the salts) will be easily visualized as wispy strands in the lysate.

MATERIALS

1 strawberry 1 50-ml tube with cap and base 1 15-ml tube with cap 1 resealable plastic bag 1 wooden stick 1 funnel

1 piece of cheesecloth 1 transfer pipette 5 ml 95% ethanol 10 ml DNA extraction buffer (detergent & salt)

PROCEDURE 1. Obtain a fresh or thawed strawberry. If you are using a fresh strawberry, remove the green sepals from the berry. 2. Place the strawberry in a resealable plastic bag. 3. Close the bag slowly, pushing all of the air out of the bag as you seal it. 4. Being careful not to break the bag, thoroughly mash the strawberry with your hands for 2 min. 5. Pour 10 ml extraction buffer into the bag with the mashed strawberry. Reseal the bag. 6. Mash the strawberry for 1 min.

Biological Principles Lab: Strawberry DNA Extraction

Name: ______________________________________ Section: _____________________________________

7. Place a funnel in a 50-ml centrifuge tube. Fold the cheesecloth in half along its longer side and place it in the funnel to create a filter. 8. Pour the strawberry mixture into the funnel, filtering the contents through the cheesecloth and into the 50-ml tube. Use the graduations on the tube to help measure the amount added. 9. Carefully pour 2 ml of the filtered contents from the 50-ml tube into a clean 15-ml tube. Use the graduations on the side of the 15-ml tube to assist you in your measurements. 10. Hold the 15-ml tube at an angle. Using a transfer pipette, carefully add 5 ml cold 95% ethanol by running it down the inside of the tube. Add the 95% ethanol until the total volume is 7 ml (use the graduations to help you measure). You should see two distinct layers. Caution: Do not mix the strawberry extract and the ethanol. 11. Watch closely as translucent strands of DNA begin to clump together where the ethanol layer meets the strawberry extract layer. Tiny bubbles in the ethanol layer will appear where the DNA precipitates. 12. Slowly and carefully rotate the wooden stick in the ethanol directly above the extract layer to wind (or "spool") the DNA. Remove the wooden stick from the tube and observe the DNA.

(Directions from Carolina Biological Supply Company - 2004)

CLEAN-UP

Discard all solutions in the sink. Return the test tubes to the instructors bench. Discard disposable pipettes, used cheesecloth, wooden sticks, and plastic bags into the Return all other materials to the instructors bench. Wipe down your lab bench with cleanser and paper towels. Push your chair in when you leave.

trash.