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INTRODUCTION: The instructions that tell a living organism how to develop, how large to grow, or what molecules to

 produce are contained in the nucleus of every cell of that organism. The cell’s

nucleus contains DNA, which stores genetic information. In eukaryotes, such as plants, DNA is

packaged into structures called chromosomes. Chromosomes contain genes, which are the units of hereditary

information. Each chromosome consists of a single large DNA molecule that contains

hundreds or thousands of different genes, depending on the organism. For example, humans have

approximately 100,000 genes. A DNA molecule is a polymer composed of many repeating sub-

units called nucleotides. Each nucleotide, in turn, is made of a 5-carbon sugar, a phosphate, and

a nitrogenous base. There are four types of bases in DNA: A,T,C, and G, which give the nucleo-

tide specific characteristics. The genetic information encoded in a DNA molecule is determined

by the sequence of nucleotides in the chain.

    Scientists have been able to increase their knowledge of the structure and function of chromo-

somes and genes, and utilize that knowledge in biotechnology, thanks to the development of

suitable techniques. One important technique has been the extraction and purification of DNA

from living organisms.

    In this laboratory exercise, we will extract and isolate the DNA contained in both onion

and cyanobacteria, which will give us an example of both prokaryotic (cyanobacteria) and

eukaryotic (onion) cells. When extracting DNA from any organism, we must break open the cell

walls and cell and nuclear membranes. Once these structural components are released from the

cells, we must take advantage of the chemical properties of all the cell contents to separate the

DNA from all the other molecules contained within the cells.

the objective of this lab is to gain hands-on experience in isolating and purifying DNA from

two different kinds of cells, and to compare and contrast them.



MATERIALS AND METHODS: DNA isolation involves three steps: 1) Homogenization, 2) De- proteination, and 3)

 Precipitation of DNA. Each of the steps requires specific chemicals,techniques, and procedures that must be carefully


 Homogenization: In this step, we will break up the onion tissues to separate and open the

cells.  Cell walls and cell and nuclear membranes get fragmented, and molecules contained

within the cells (including the DNA) are liberated into the medium. We will briefly heat the

tissue before homogenization in order to inactivate DNAases (enzymes that degrade DNA)

that are present in our mixture. Once we degrade DNAases, we will maintain the homogenate

at a cold temperature in order to keep the enzymes deactivated and the DNA cold for the

precipitation step.  After we homogenize the onion tissue in the blender, we will strain it

through cheesecloth to remove the bigger debris such as cell walls, membranes, etc.

Cut a medium sized onion into 2-3mm segments.

Weigh 50g. of onion pieces and transfer them to a 250ml beaker.

Add 100ml homogenizing medium (buffer) to the beaker and incubate in a 65C-water bath for 15 minutes.

Immediately place the beaker in an ice water bath (15-20C) for 10-15 minutes.

Pour the suspension into a blender and homogenize for 30-40 seconds at low speed, and then 15-20 seconds at high speed.

Pour the homogenate back into the beaker and place in an ice bath for 5-10 minutes.

Filter the homogenate through 4 layers of cheesecloth into a 250ml beaker. (Make sure the foam does not go into the beaker).

Deproteinization: In this step, we will purify the homogenate by removing proteins and lipids

 that are associated with cells and DNA molecules. We will take advantage of differential

 solubility of DNA, proteins, lipids, and other macromolecules to separate them using different

 solvents. While DNA is soluble in water, other macromolecules are more soluble in organic 

 solvents, such as chloroform (some protocols will call for other organic solvents if a fume

 hood is not available). If we add chloroform to our homogenate (which contains water), two

 layers will form. Those molecules that are soluble in water will remain in the top water layer,

 and those molecule that are chloroform-soluble will drop to the bottom chloroform layer.

Pour 80ml of ice-cold 95% ethanol into a 250ml flask and place in the ultra-cold freezer at-80C (the instructor will do this for you). It will be used in the precipitation step of the procedure.

Be sure that you have at least 50ml of the filtered homogenate in the 250ml flask.

Use a 5ml pipette to add 2ml of chloroform to the homogenate. Pour the chloroform gently down the side of the flask (watch the instructor demonstrate the procedure first). Two layers will form in your flask.

Gently swirl the flask—if you mix the layers too much, an emulsion will form and the layers will disappear.

Pour off the top aqueous layer into a clean 250ml flask very gently. You don’t need to get ALL of the top layer, just most of it. Discard the bottom chloroform layer into the Chloroform Waste Container.

Repeat steps 3 thru 5 one more time. (Add chloroform to the beaker containing the aqueous layer that you obtained, swirl, pour top layer into a clean flask, and discard the chloroform layer).

Pour the bottom chloroform layer, which contains all the undesired proteins, into the Chloroform Waste Containe

Precipitation of DNA: In this step we will precipitate the DNA (force it to come out of solution) by using very cold alcohol (placed earlier in the ultra-cold freezer). When the precipitation is completed, the liquid within the flask will become cloudy. This cloudiness is really the long nucleotide polymer---DNA! Once we are at this step we will be able to spool the long DNA strands onto a very cold glass rod and out of the beaker.

Very slowly pour the ice- cold ethanol down the side of the flask so that it sits on top of the homogenate without disturbing the layer.

Very slowly begin to twirl an ice-cold glass-stirring rod that has been specially bent for DNA retrieval. DNA strands should wind around the glass rod. Be very gentle or the strands of DNA will break and you will not be able to spool it.

RESULTS: After having completed the above procedure, a long, thick, white, mucus-like substance

wrapped itself around the special stirring rod. This was the DNA. The instructor told us

that this was a very good yield for the amount of onion that was used. The DNA was added

to a preservative solution to be used for future experimentation.



The following procedure is very similar to the onion isolation and purification. There-

fore, only the protocol steps will be outlined, and the student can make the connections to the

reasons behind the steps if they are not explained in the protocol.

Cell lysis:

Add 1.0ml cell suspension (e.g., overnight culture containing approximately 1-3 billion cells) to a 1.5ml tube on ice.

Centrifuge at 13,000-16,000-x g for up to 5-60 seconds to pellet the cells. For some species centrifugation for up to 60 seconds may be required to obtain a tight pellet. Remove as much supernatant as possible using a pipet.

Add 600ul Cell Suspension Solution to cell pellet and gently pipet up and down until cells are suspended. (This is a buffer to prevent denaturation of the DNA).

Add 3.0ul Lytic Enzyme Solution and invert tube 25 times to mix.

Incubate at 37C for 30 minutes to digest cell walls. Invert sample occasionally during the incubation.

Centrifuge at 13,000-16,000-x g for 1 minute to pellet the cells. Remove supernatant.

Add 600ul Cell Lysis Solution to the cell pellet and gently pipet up and down to lyse the cells.

For some species, heating the sample to 80C for five minutes may be required to complete cell lysis. Do this.

RNAase Treatment:

Add 3.0ul RNAase A Solution to the cell lysate.

Mix the sample by inverting the tube 25 times and incubate at 37C for 15-20 minutes.

Protein Precipitation:

Cool the sample to room temperature (place in a beaker with room temperature water).

Add 200ul Protein Precipitation Solution to the cell lysate.

Vortex vigorously at high speed for 20 seconds to mix the Protein Precipitation Solution uniformly with the cell lysate. For species with a high polysaccharide content, placing the sample on ice for 15-20 minutes may be required. For this cyanobacterium, use 20 minutes.

Centrifuge at 13,000-16,000-x g for 3 minutes. The precipitated proteins will form a tight white pellet. If the protein pellet in not tight, repeat Step 3 followed by incubation on ice for 5 minutes, then repeat Step 4. Save the supernatant.

DNA Precipitation:

Pour the supernatant containing the DNA (leaving behind the precipitated protein pellet) into

a clean 1.5ml microfuge tube containing 600ul 100% Isopropanol (2-propanol).

Mix the sample by inverting gently 50 times.

Centrifuge at 13,000-16,000-x g for 1 minute; the DNA should be visible as a small white pellet. (Show this to the instructor for verification).

Pour off supernatant and drain the tube briefly on clean absorbent paper. Use a Chemwipe to remove any residual drops of alcohol. Add 600ul 70% Ethanol and invert the tube several times to wash the DNA pellet.

Centrifuge at 13,000-16,000-x g for 1 minute. Carefully pour off the ethanol.

Invert and drain the tube on clean absorbent paper and allow to air dry for 15 minutes.

DNA Hydration:

1. Add 200ul DNA Hydration Solution (Tris-EDTA). (50-100ul will give a concentration of

100ug/ml if the yield is 20 ug DNA).

Allow the DNA to rehydrate overnight at room temperature. Alternatively, heat at 65C for

1 hour. Tap tube periodically to aid in dispersing the DNA. Store DNA at 2-8C.

The above protocol was taken from Puregene DNA Isolation Kit, Gentra Systems, Inc.

RESULTS: The pellet was collected and set aside for use in further experimentation—PCR

Amplification of DNA, Agarose Gel Electrophoresis, Ligation and Transformation, and

Restriction Endonuclease Digestion. Students should record their observations and results below

and answer the following questions.

What are the color and consistency, texture, etc. of the DNA you extracted? Be sure to note any differences between the onion and the cyanobacterial DNA.

Opaque white, mucus-like, slimy texture, etc. No differences could be seen.

What would happen if we didn’t do steps 3 and 4 of the homogenizing procedure?

Buffering prevents nucleases from attacking DNA. Cold water keeps DNA intact

and enzymes (nucleases) deactivated. The DNA would have denatured.

As a scientist doing this procedure in his/her lab, what could you do with the DNA

you obtained?

Further experimentation, such as polymerase chain reaction, restriction enzyme

mapping, try to create an onion or cyanobacterial cell "from scratch", create a

DNA library, etc.

DISCUSSION: The extraction of DNA from the nucleus of onion cells and from prokaryotic cyano-

bacterial cells is an excellent illustration of how far scientists have come in identifying, isolating,

and further working with DNA. The students will hopefully realize that this

field of study is just opening up to them, and there are hundreds of related

jobs out there for their choosing. The students who begin this series of

experiments will also feel that they are "on the cutting edge"of scientific

discovery. Hopefully they will supplement this experiment with readings and

noting current events that are related.

CONCLUSION: At the completion of this lab, the students should feel more confident about

their knowledge of cellular characteristics, chemical effects on and methods to eliminate

cell parts to liberate nuclear contents, and laboratory technique and manipulation of lab tools.

Their subsequent use of the DNA collected in this lab also should help them build connections

from one procedure to the next, and realize that the completion of one exercise is not the end of

the work. It should help "build connections."


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