DNA Extraction from Wheat Germ - Download as DOC

Document Sample
DNA Extraction from Wheat Germ - Download as DOC Powered By Docstoc
					                                                        Name:

                                                        Period:    Date:

        DNA extraction is fundamental procedure in scientific laboratories around the
world. By extracting DNA and studying just it alone (separated and purified from the
other cell parts and substances), scientists can learn how DNA encodes the instructions
for all life processes.


        DNA of course is important in the study of heredity (passing of traits through
genetic material generation to generation) and the treatment of all genetic diseases. It
can be for identification through DNA fingerprinting (matching patterns of the bases).
Scientists can genetically engineer changes in the DNA sequence of the genes to modify
(change) the traits of various plant, animal, bacterial species—any form of life even
viruses.


        All life forms and even most viruses have DNA as their genetic
material they inherit from the previous generation. On the ‘code of
life’ can be found the designs that control what our cells become and
how they function and their life cycle. Multicellular organisms have
different cell types. Each cell types only use the code from genes for their specific cell
type even though every body cell contains approximately 25,0000 genes located over 23
pairs of chromosomes in their nucleus.


        This lab is designed to extract design DNA from any DNA source (fresh tissue).

Some questions to get you thinking about the lab:

   1.   One way to purify a molecule is to get rid of everything but that molecule. If we
        want to isolate DNA from cells, what do we have to get rid of?




   2.   What materials would you use to do that?
                                 Instructions




1. Place 50 grams or 1 cup of cell source
  into the blender container. Add 60 ml
  warm water with 1 gram of salt. The trick is not to add too much water—the
  mixture should be like thick soup or opaque like chocolate milk. Place the lid on
  the blender and make sure that it is secure. Blend at low for 30 seconds to start.
  Blend at liquefy for 10 seconds, pause, check,
  and agitate. Repeat 2 times or until it is
  thoroughly liquefied.


  Pour the liquefied cell solution through two
  layers of cheese cloth covering a funnel into a
  clean 50 ml tube. When the test tube is about 1/4 full, take the funnel out of the
  test tube and discard any extra mashed pulp, and the cheesecloth. This filters the
  liquid part of the cell from more solid portion.



                                 2. Add a pinch of a meat tenderizer and 5 ml of
                                    liquid soap. Invert the tube to mix the contents,
                                    but do not shake it or stir very gently, trying
                                    not to make bubbles. Do this a few times.




3. Then let it sit in hot (50-60 °C) tap water bath for about 15 minutes. Do not use
  water hotter than 50-60° C.-it will destroy the DNA. The water will cool during the
  extraction procedure, but this does not matter. Test your tap water -- it may be
  hot enough right from the tap.
Why use a blender?

The blender helps in the process of not only liquefying the tissues of the plant or animal
sample but also in breaking open the cells themselves (called cell lysis) to access the
DNA inside the nucleus.
Why is water temperature important?

The heat softens the phospholipids (fats) in the membranes that surround the cell and
the nucleus. It also inactivates (denatures) the deoxyribonuclease enzymes (DNase)
which, if present, would cut the DNA into such small fragments that it would not be
visible. Denatured enzymes and DNA unravel, lose their shape, and thus become
inactive. Enzymes denature at 60° Celsius, and DNA denatures (falls apart) at 80°
Celsius.
Why is salt needed?

The salt in the solution will help neutralize the negative charges on the DNA molecules
so they will precipitate (separate and solidify and become visible).

Why use meat tenderizer?

Actually you don’t usually, unless your source of DNA is animal tissue. It has enzymes
that will release the DNA molecule from the proteins (round shaped histone proteins)) it
is attached to. Pineapples, strawberries, onions, and kiwi fruit (among others) naturally
contain their own protease (means ‘protein cutting’) enzymes. The adding of enzymes is
optional according to most procedures.
Detergent/Soap

Detergent contains sodium laurel sulfate,
which cleans dishes by removing fats
and proteins. It acts the same way in the
DNA extraction, pulling apart the fats
(lipids) and proteins that make up the
membranes surrounding the cell and
nucleus. Once these membranes are broken apart, the DNA is released from the cell.
Soap molecules and grease molecules                                                   are
made of two parts:
      Heads, which like water
      Tails, which hate water.
Both soap and grease molecules
organize themselves in bubbles
(spheres) with heads outside to face the water and tails inside to hide from the water.

When soap comes close to grease, it captures it, forming a greasy soapy ball.
A cell's membranes have two layers of phospholipids (fat) molecules with proteins going
through them.
When detergent comes close to the
cell, it captures the lipids and
proteins breaking (lysing) the cell
and nuclear membranes and
releases the DNA.


   4. Pour about 2-5 mls of the cell solution from the 50 ml tube into a 15 ml tube. Use
      an eyedropper, pipette, or piece of paper towel to remove any foam from the top
      of the solution.


   5. Tilt the 15 ml tube at an
      angle. SLOWLY pour 5 ml of
      icy cold alcohol (ethanol)
      down the side so that it forms
      a layer on top of the cell
      solution. Do not mix the two
      layers together. DNA separates
      (precipitates) at the water-
      alcohol interface (the boundary
      between the water and the
      alcohol). Therefore, it is crucial
      to pour the alcohol very
      slowly so that it forms a layer on
      top of the water solution. If the
      alcohol mixes with the water, it will become too dilute and the DNA will not
      precipitate.
                 Let the test tube, beaker or jar sit for several minutes. White, stringy,
      filmy DNA will begin to appear where the water and alcohol meet. You will usually
      see DNA precipitating from the solution at the water-alcohol interface as soon as
      you pour in the alcohol. If you let the preparation sit for 15 minutes or so, the
      DNA will float to the top of the alcohol.
Why use concentrated alcohol?

Watch carefully as the DNA precipitates or separates and moves through the alcohol
layer with small bubbles attached to the strands. The DNA released from the cell nucleus
is dissolved in the water/detergent/wheat germ solution and cannot be seen. DNA
precipitates out of solution in alcohol, where it can be seen as white mucous like
strands. Besides allowing us to see the DNA, the alcohol separates the DNA from the
other cell components, which are left behind in the water solution. You should use 70-
95% isopropyl, ethyl alcohol, or methylated spirits –that is very concentrated (less
water). Why cold ethanol? The cold temperature keeps the DNA from breaking apart
(dissolving) so easily. It is very thin and fragile.


   6. Use a wooden stick or plastic or glass rod to collect (spool) the DNA. Insert the
      wooden splint into the 15-mL tube until its tip is just below the interface of the two
      layers. Turn and twist the splint/rod. Do not mix the two layers together. The
      DNA should wrap around the splint like yarn on a spool.


   7. If you want to keep the DNA, store it in 50 - 70% alcohol in a sealed tube or air
      dry it on paper towels or filter paper.


   8. Observe and analyze the threads of DNA and then answer the following questions.




Now that you've successfully extracted DNA from one source, you're ready to experiment
further. Try these ideas or some of your own (of course you should get parent permission):

Experiment with other DNA sources. Which source gives you the most DNA? How can you
compare them?

Experiment with different soaps and detergents. Do powdered soaps work as well as liquid
detergents? How about shampoo or body scrub?

Experiment with leaving out or changing steps. We've told you that you need each step, but is
this true? Find out for yourself. Try leaving out a step or changing how much of each ingredient
you use.

Do only living organisms contain DNA? Try extracting DNA from things that you think might not
have DNA.
DNA Extraction Lab Name:                           Period:      Date:
Observations:   Describe what the strawberry DNA that you pulled out of the test tube looks like.



Questions
1.    Give two reasons why a scientist might want to study DNA?



2.    What part of the above procedure will break open the cell walls? _____________

3.    Which part of the above procedure will break open the cell membranes?
      ______________________

4.    What part of cells would be most affected the soap molecules? ______________


5.    When you mix the blended cell source with the soap, what is happening?

6.    What does the salt do?


7.    What does the alcohol do? Why does the DNA rise to the top after adding alcohol?



8.    Why can’t you see the double helix? What form of DNA can you see with a basic
      Light microscope?


9.    What part of the cell did the DNA come from? ________________

10.   What is the pH of DNA? (Actually there is some RNA in the precipitate too).
      __________ pH
11.   What substance (organic molecule) is most affected by the enzymes
      in the meat tenderizer? ____________________

12.   Would DNA be the same in all of the cells in your body? (Hint: Remember that
      each person started out as one fertilized cell that just kept on going through the
      process of mitosis to create the person you are today)

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:32
posted:2/25/2012
language:English
pages:6