“MASTER EXTRACTOR”DNA ISOLATION LAB

Purpose: To obtain firsthand experience with DNA by isolating it from plant tissue.

Objectives: Become familiar with the physical properties of DNA by isolating it fromliving tissue. Explain the purpose

of each step in the isolation process as itrelates to thephysical and biochemical characteristics of thegeneticmaterial.

Background Info: To most people, DNA is abstract. The history of DNA’s discovery can be learned, and thestructures of DNA can be memorized, but until you actually handle DNA, it can remain a mysterious and unknown substance. In this lab, you will have the opportunity to actually see the DNA of a plant cell. The DNA of a cell can be extracted from the nucleus once the cellular boundaries are broken down. We’ll use bananas as the source of plant cell DNA. 

DNA is covered in proteins known as histones and coils tightly around them. In order to isolate DNA, the cells must be broken open (boundaries broken down) and the proteins surrounding DNA must be denatured. This is done in a series of steps. Thefirst step, which is to break down the cell wall (if present), cell membrane, and nuclear envelope, is accomplished by a process called homogenization. You will homogenize the bananaby using a zip lock baggie. In this set-up, a buffer solution is used to complete the homogenization process by denaturing the histones that surround the DNA. The next step of this extraction process is accomplished by adding ice cold ethanol to the homogenate. DNA is insoluble in ethanol and will precipitate out at the interface of the two liquids. If this step is performed with great care using proper technique, you should be able to extract the fine, long strands of DNA by using a glass stirring rod or wood splint to “spool” out the DNA.

Materials:

Bananatest tubeZiploc baggie95% ethanol

Funnelcoffee filterglass stir rodpipettes

Buffer solutiongraduated cylinder

Procedure:

1. Obtain a slice of banana and place it in the zip-lock baggie.

2. Add 7 ml of water, and 3 ml of DNA buffer to the baggie. Remove as muchair asyou can from the baggieand then seal

the baggie very well.

3. Carefully and gently pulverize the banana using your hands until the banana iscompletelymashed. Make sure that

the buffer solution and water are mixed in wellwith the mashed upbanana. Continue pulverizing for 3 minutes.

4. After 3 minutes, filter the solution through a coffee filter (placed into the funnel) intothetest tube. When filtering is

complete, discard thebaggie and usedcoffee filter.

5. Carry the test tube to the vent hood. Carefully tilt the vial and add two pipettes full of icecold ethanol down the side of

the vial. The ethanol should form a layer across the topof the bananasolution if it is added correctly.

6. A precipitate will form at the interface. Use a glass stir rod to spool out the DNA. Place thestir rod upright in the

test tube, allowing the tip to touch the bottom of the vial. Slowly turn theglass stir rod continuously in the same direction.

You are trying towind the DNA on the rod,not stir the solution. If you have successfully spooled outthe DNA, you

have earned the title– MASTER EXTRACTOR!!

7. When you are done, dump all contents down the drain and rinse your test tube out in the sink. Clean and dry your lab

station thoroughly!!! Answer the analysis questions on a sheet of paper incompletesentences!

Analysis Questions:Please answer in complete sentences!!

1. Describe the appearance of the DNA that you isolated in the bananasolution. (answer scientifically using qualitative data)

2. What is the monomer of DNA? What is each monomer composed of?

3. What type of bonds hold the components of each nucleotide together? What type ofbondforms between thehydrogen

bases of DNA?

4. Explain Chargaff’s findings through his experimentation. How are these findingsimportant toDNA replication?

5. Specifically, in what part of the cell cycle does replication occur? Where does thisreplicationoccur in the cell?

6. The sequence of bases on one strand of DNA is GGCAGTTCATGC. What would bethesequence on the

complementary strand of DNA?

7. If you were extracting DNA from a bacterial cell, which boundaries would you havetoremove? Which boundaries

would be missing?