Lab Exercise 10 - ISOLATION OF DNA FROMPLANTS

Introduction

Deoxyribonucleic acid (DNA) is located in thenucleus of eukaryotic cells (animals, plants,fungi, and protists). DNA contains information to direct the cellin the manufacture of proteins. Proteins controldevelopment, organ function, metabolism, enzymatic reactions,photosynthesis, muscle action, brain activity, and many othercellular processes. DNA is often referred to as the “blueprint forlife”.

DNA is a polymer composed of the nucleotidebases guanine (G), adenine (A), thymine (T), and cytosine(C), and two sugar/phosphate backbones. Two DNA strands are twistedto form a double helix. The number of nucleotidebases (G, A, T, C) in each human cell is about 3 billion. The 3billion base pairs in the human genome are locatedon 46 strands of DNA called chromosomes. The HumanGenome Project has determined the order of the nucleotides on eachchromosome. A gene is a sequence of nucleotidebases (DNA) that codes for a specific protein. Human DNA containsabout 20,000 genes while the cells of the rice plant contain over40,000 genes.

In the DNA isolation procedure, plant cell walls and cellmembranes are broken down by blending or mashing and heating thecells. Detergent in the extraction solution dissolves lipids in thecell membrane causing the cells to lyse. Whencells undergo lysis, the cellular components, including the DNA,are released. The technique of filtration uses amedium, in this case cheesecloth, to separate solids from liquids.The resultant material is referred to as filtrate.When cold ethanol is added to the filtrate, DNAprecipitates at the water/ethanolinterface. Although an individual DNA molecule isnot visible with the naked eye, DNA isolated from large quantitiesof cells can be observed.

Procedure: Isolation of DNA from plants

Strawberry seeds also contain large amounts of DNA, a commonlycultivated strawberry, Fragaria ananassa, is octoploid –contains eight sets of chromosomes in each cell.

Materials

                                                                                                 

*Strawberries

*Ice in tray

fork                

100 ml beaker

Scissors

95% Ethyl alcohol, ice cold

Plasticbag                                                      

PART ONE: Lyse plant cells

                                   

1. Obtain one medium size strawberry. Frozen strawberries seemto work best. Place the strawberry into the plastic bag, use a forkor prees to mash the strawberry thoroughly. Place the mashedstrawberry into a 250 ml beaker

3. Use the graduated cylinder to measure 30 ml extraction bufferthat has been pre-heated to 60^oC.

4. Swirl mixtures constantly for 5 minutes.

Questions

What occurs to cell membranes when exposed to detergent in theDNA extraction?

Describe cell lysis.

PART TWO: Filter plant cellextract

Obtain a pieces of double-layer cheesecloth and a 50 ml beakers.Place a piece of cheesecloth over the conical tube, you may use therubber band to secure the cheese cloth. Carefully filter strawberryuntil about 10 ml have been obtained.

Discard the cheesecloth containing the solid plant remnants(regular trash).

Questions

List 3 molecules that have passed through the cheesecloth intothe filtrate

What molecular basis does filtration select - charge,solubility, size, density?

Where is the plant DNA now located?

PART THREE: PrecipitateDNA:

1. Use a graduated cylinder to measure (20 ml) ice-cold ethanol.Slowly pour the ethanol down the side of theconical tube so that the ethanol is layered over the filtrate. DONOT MIX.

2. Let the preparation sit undisturbed for up to 5 minutes.Observe the interface.

At this point, PHOTOGRAPH YOUR WORK. Submit the imagealong with this worksheet to the assignment drop box in thecourse.

PART FOUR: Spool DNA

Spool DNA from the conical tube onto a wooden rod. Obtain asmuch DNA as possible on the rod. Lift the DNA out of the solutionand blot the excess alcohol on a paper towel.

Use a wooden rod to spool the DNA as demonstrated by theinstructor.

Describe the appearance of the DNA interms of color and substance

What is the role of ethanol in theDNA extraction procedure?

Record Observations (the appearance of DNA andother observations during spooling)