Question

Lab Exercise 10 - ISOLATION OF DNA FROM PLANTS

Introduction

Deoxyribonucleic acid (DNA) is located in the nucleus of eukaryotic cells (animals, plants, fungi, and protists). DNA contains information to direct the cell in the manufacture of proteins. Proteins control development, organ function, metabolism, enzymatic reactions, photosynthesis, muscle action, brain activity, and many other cellular processes. DNA is often referred to as the “blueprint for life”.

DNA is a polymer composed of the nucleotide bases guanine (G), adenine (A), thymine (T), and cytosine (C), and two sugar/phosphate backbones. Two DNA strands are twisted to form a double helix. The number of nucleotide bases (G, A, T, C) in each human cell is about 3 billion. The 3 billion base pairs in the human genome are located on 46 strands of DNA called chromosomes. The Human Genome Project has determined the order of the nucleotides on each chromosome. A gene is a sequence of nucleotide bases (DNA) that codes for a specific protein. Human DNA contains about 20,000 genes while the cells of the rice plant contain over 40,000 genes.

In the DNA isolation procedure, plant cell walls and cell membranes are broken down by blending or mashing and heating the cells. Detergent in the extraction solution dissolves lipids in the cell membrane causing the cells to lyse. When cells undergo lysis, the cellular components, including the DNA, are released. The technique of filtration uses a medium, 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, DNA precipitates at the water/ethanol interface. Although an individual DNA molecule is not visible with the naked eye, DNA isolated from large quantities of cells can be observed.

Procedure: Isolation of DNA from plants

Strawberry seeds also contain large amounts of DNA, a commonly cultivated 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

Plastic bag                                                      

PART ONE: Lyse plant cells

                                   

1. Obtain one medium size strawberry. Frozen strawberries seem to work best. Place the strawberry into the plastic bag, use a fork or prees to mash the strawberry thoroughly. Place the mashed strawberry into a 250 ml beaker

3. Use the graduated cylinder to measure 30 ml extraction buffer that 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 the DNA extraction?

Describe cell lysis.

PART TWO: Filter plant cell extract

Obtain a pieces of double-layer cheesecloth and a 50 ml beakers. Place a piece of cheesecloth over the conical tube, you may use the rubber band to secure the cheese cloth. Carefully filter strawberry until 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 into the filtrate

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

Where is the plant DNA now located?

PART THREE: Precipitate DNA:

1. Use a graduated cylinder to measure (20 ml) ice-cold ethanol. Slowly pour the ethanol down the side of the conical tube so that the ethanol is layered over the filtrate. DO NOT MIX.

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

At this point, PHOTOGRAPH YOUR WORK. Submit the image along with this worksheet to the assignment drop box in the course.

PART FOUR: Spool DNA

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

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

Describe the appearance of the DNA in terms of color and substance

What is the role of ethanol in the DNA extraction procedure?

Record Observations (the appearance of DNA and other observations during spooling)

Answer 1

PART:ONE

ANSWER 1. Cell membrane or plasma membrane is composed of lipids and proteins. So, when detergents are added they emulsify the lipids and break the proteins of the plasma membrane and the nuclear membrane so that DNA can be freed from the nucleus and easily extracted.

Answer 2. Breaking of the cell and releasing of the cellular contents is known as cell lysis.

PART:TWO

ANSWER 1. Chromatin, DNA, ribosomes can cross the cheese cloth. Chromatin has genes on it that contains DNA.

Answer 2. The filteration process filters the molecules basing upon their SIZE.

Answer 3. The plant DNA is now present in the filtrate. It can be extracted by certain chemicals.

PART THREE

Answer 1. DNA appears as white, strinky mass.

Answer 2. DNA is insoluble in the ethanol, thus it forms white strinky mass in seconds and cen be easily separated.

Record observations:

1. After adding ethanol to the filtrate wait for 30 seconds or so.

2. DNA will form a white strinky mass at the supernatant position in the filtrate.

3. This DNA can be carefully separated with the help of glass rod through spooling process and kept in another small tube for further analysis.

4. Spooling picks a good mass of DNA in a single attempt.