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Protocol for DNA Extraction from Bananas DNA Extraction from Tomatoes

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Protocol for DNA Extraction from Bananas DNA Extraction from Tomatoes Powered By Docstoc
					DNA Extraction from Tomatoes
DNA is the genetic information inside of cells. Every living organism is
comprised of cells has DNA that provides the instructions for making proteins
needed for survival. When specific sections of DNA called genes are turned on,
gene expression is said to occur. As different genes are expressed, different
characteristics appear in the cells and the organism as a whole. The process of
turning genes on during gene expression first involves                          http://www.graphic-
production of RNA molecules and then production of                              design.com/Web/Critique/tomato.html

proteins that act as enzymes, messenger molecules
(hormones), and structural components within the cell and
organism as a whole.

Think about tomatoes. A fresh ripe juicy summer tomato stimulates your taste buds in all the right places.
But in the winter tomatoes tend to be tougher and tasteless. Both types of tomatoes are fruits that come
from the same species of tomato plant. Because they are members of the same species, they have the same
amount of DNA and the same types of genes although it is possible the genes have different alleles. The
summer tomato is ripe and sweet, while winter tomato is not fully ripe and has not had the opportunity to
develop the same flavor characteristics as the summer tomato. These differences are due to gene
expression. While the DNA in both tomatoes is the same, the summer (fully ripe) tomato has genes being
expressed that lead to sugar production for sweetness, red pigments for coloration, and enzymes for
destruction of green chlorophyll. The longer the fruit is on the plant, the more fully the genes are
expressed. Gene expression within cellular DNA is important for differentiation (specialization of cells)
in every multicellular organism. As well, single cellular organisms may use gene expression to turn genes
on and off to match specific environmental conditions around them.




Materials:
Fresh tomatoes (1 wedge/ group about 3cm at widest point)
Extraction Solution: Salt, Soap (shampoo), water (20ml per group)
Ziploc Bags (1 per group)
Cheesecloth or #2 coffee filter (1 per group)
Styrofoam Cup (1 per group)
50 mL Tubes or Beakers (1 for each group)
10 mL Graduated Cylinder (1 per group)
Cold Alcohol (5-10ml per group)                           http://www.eatcatomatoes.org/
3x5 Card (1 per Group)                                    Consumer/Content.aspx?id=62

Methylene Blue (2-3 Drops)




                                                                  www.dnamnd.med.usyd.edu.au/DNA_logo.jpg
Procedure:

1. Place tomato piece in a Ziploc Bag

2. Add your 20mL of the Extraction Solution to the Ziploc bag. Carefully remove as much air as possible
without spilling the liquid out of the bag and zip the bag closed. Squeeze the tomato pieces and solution
inside the bag with your fingers or beat the tomato pieces with the bottom of a blunt object for about 5
minutes. Be sure not to puncture or rip the bag during the process.

   Questions - Place your answers on the student answer sheet.

      What does squeezing or beating do to the cells that make up the tomato?
      The Extraction Solution in the bag is made up of soap, salt, and water. What do these ingredients
       do to the cells that make up the tomato? Remember the pre-lab…
      Take a small amount of the tomato and Extraction solution mixture and prepare a wet-mount slide.
       Observe on low power then switch to high power and draw cells after they have been lysed.
      What is lysis?

3. Filter the mixture through the cheesecloth or coffee filter. Be careful to allow only the liquid and not
chunks of tomato that remain into the Styrofoam cup.

   Question - Place your answers on the student answer sheet.

      Is the tomato cell DNA being trapped in the filter or moving through the filter in this step of the
       procedure?

4. Measure 10 mL of the filtered tomato solution using a graduated cylinder and place in tube or beaker.

5. Add 10 mL of cold alcohol to the 50 mL tube with the tomato solution in it and start timing. Do not
shake or swirl the tube. Watch the tube closely and observe the DNA as it comes out of the solution and
forms a condensed mass within the tube. Record the changes that occur inside of the tube and the amount
of time it takes for each change to occur.

   Questions and Observations - Place your answers on the student answer sheet.

      Describe what happened to the DNA solution when the cold alcohol was added. In doing so,
       accurately describe the changes that occurred and give the amount of time it took for each change
       to occur.
      If the alcohol had not been cold, do you suppose the changes in the tube would have occurred
       more slowly or more quickly? State your hypothesis and give a reason for your idea.

6. Using a plastic spoon, carefully scoop the DNA out of the tube or beaker and place it on a paper 3x5
card. Add 2 or 3 drops of methylene blue to completely cover the DNA and let the DNA sit undisturbed
overnight.
    Questions - Place your answers on the student answer sheet.
      Did the DNA on the card stain blue? _________
      What parts of the tomato cells stained blue on the wet-mount prepared in the pre lab? How dies
       this correspond to the stained DNA?
DNA Extraction - Student Answer Sheet                                Name ______________________


Questions from Step 2

      What does squeezing or beating do to the cells that make up the tomato?




      The Extraction Solution in the bag is made up of soap, salt, and water. What do these ingredients
       do to the cells that make up the tomato?




      Drawing:




      What is lysis?


Question from Step 3

      Is the tomato cell DNA being trapped in the filter or moving through the filter in this step of the
       procedure?




Questions and Observations from Step 5

      Describe what happened to the DNA solution when the cold alcohol was added. In doing so,
       accurately describe the changes that occurred and give the amount of time it took for each change
       to occur.
      If the alcohol had not been cold, do you suppose the changes in the tube would have occurred
       more slowly or more quickly? State your hypothesis and give a reason for your idea.

      Record the changes that occur inside of the tube and the amount of time it takes for each change to
       occur.




Questions from Step 6


      Did the DNA on the card stain blue? _________

      What parts of the tomato cells stained blue on the wet-mount prepared in the pre lab? How does
       this correspond to the stained DNA?




Summary Questions

   1. Make a list of descriptive words that could be used to explain what DNA looks like to someone
      who has never seen DNA before.




   2. If you could repeat this experiment several times, what other substances would you choose to try
      and get DNA? Explain your reasoning and thinking.
Teacher Notes
Tubes

Corning - Cat #430897 - 50mL Centrifuge Tube, Self-Standing – Case of 500)

Corning® 50 mL Centrifuge Tubes



Extraction Solution (500mL)

Mix 50 mL of Shampoo and 7.5g Salt. Dissolve salt by stirring slowly to avoid bubbles/foaming. Add
water to a final volume of 500 mL. Measure 20ml per group.

Note1: Do not use shampoos with conditioner or baby shampoo.
Note2: Suave and White Rain shampoo work well.

The detergent dissolves the lipids that comprise the cell membranes. After cellular membranes are
disrupted, the DNA and cell components are present in the solution. The detergent causes lipids and some
proteins to precipitate out of solution, leaving DNA and some proteins.

The salt will ionize in solution to form Na + and Cl- ions. These ions bind with DNase enzymes that will
degrade the DNA and serve to help protect the DNA during the extraction process. As well, Na + will
interact with the negatively charged regions on the DNA so that strands of DNA will come together
during the extraction process.


Alcohol - Cold 95% Ethanol or Isopropanol (rubbing alcohol) – Place in Freezer Prior to Use

The alcohol acts to remove water from around the DNA. Alcohol forces the water away from the DNA
and causes it to precipitate into a wad.

The alcohol must be cold for optimal results, otherwise the alcohol and water will mix more readily and
the water will not be drawn away from the DNA. As a result, the DNA will not precipitate.


Methylene Blue

Methylene Blue stains the Nucleus and Cytoplasmic Granules of cells blue and will stain the DNA
extracted from tomatos blue.

Hydrophilic Molecule – Basic (+ charge) will associate with acidic R-groups of protein molecules and the
sugar phosphate backbone of DNA (- charge). Methylene blue will also intercalate with the base pairs in
the inner region of the DNA since it is a 3-ringed flat molecule. As well, methylene blue will turn
colorless when reduced and can be used as an indicator for REDOX reactions - peak absorbance at
wavelengths of 609nm and 668nm when blue.

Preparation - Mix 1g in 25ml of dH2O. Note: If mixed in ethyl alcohol, it will dehydrate DNA.

Extensions – Other Staining Procedures

Iodine – Stains the Nucleus, Cilia, and Flagella of Cells

Iodine will not stain the DNA. It will remain brown/amber and not change to purple, blue, or black. This
shows that there is no starch (polysaccharide) associated with the DNA.


Acetocarmine – Stains the Nucleus / Chromosomes / DNA

Acetocarmine will stain the DNA red and is a common stain for the nucleus cells. Acetocarmine is a basic
dye with a positive charge that is attracted to the negative charge of the DNA. Note the precautions on the
acetocarmine stain and use appropriate lab safety precautions.


Bradford Reagent – Indicator molecules conformationally change when exposed to protein. The shift in
structure changes the wavelengths of light absorbed and reflected by the molecule. As a result, it shifts
from grayish-blue to bright blue.

Bradford Reagent
100mg Coomassie Brilliant Blue G-250
Dissolve in 50ml of 95% Ethanol
Add 100ml Phosphoric Acid (85% w/v)
Dilute with Distilled Water to a final volume of 1 Liter.

Note – Be sure to get Coomassie Brilliant Blue G –250, not R-250.

Aldrich Chemical Co.
PO Box 355
Milwaukee, WI 53201
(414) 273 – 3850

Bradford reagent stains proteins and turns from grayish blue to bright blue. All eukaryotic DNA will stain
blue with this reagent since it is associated with histone proteins. This can be a valuable technique to see
that eukaryotic DNA is actually comprised of both DNA and protein. Ask students if DNA from a
prokaryotic cell (no histones) would also stain blue with Bradford reagent.

				
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