DNA Isolation Lab
DNA, or deoxyribonucleic acid, is the set of molecular instructions for building and running any organism. It is the genetic information to direct all cell activities—making proteins. You are basically built of, and run, by proteins! DNA, for the most part, makes up the chromosomes of cells of all living things. It can be found in the nucleus of most living things. Watson and Crick were the first scientists to describe the structure of DNA as a double helix. In this lab, we will be attempting to extract DNA from various sources and then isolating it so that we can observe the individual strands of DNA.
Background Information:
There are four basic steps to extracting (taking out) DNA.
a. The cell must be lysed (broken open) to release the nucleus.
b. The nucleus (if present) must also be opened to release DNA.
c. At this point the DNA must be protected from enzymes (special proteins) that will damage or destroy it, causing shearing.
d. Once the DNA is released, it must then be transformed from liquid to a solid form. This is called precipitation and is done through the use an alcohol.
In order for the cell to be lysed, the phospholipid (fatty) molecules in the cell and nuclear membranes must be broken down. A detergent, salt solution and shaking accomplish this. Remember the commercial, “Dawn takes grease out of the way.” Heat could be used. However, it would destroy the enzymes, as well as the DNA--that is why a detergent and a salt solution are used. The prepared solution uses Epsom salts and buffered aspirin to deactivate the enzymes that degrade (break down) the DNA when released; the solutions also stabilize the DNA. Remember that DNA is supposed to stay in the nucleus. Enzymes will break down DNA if it is detected outside the nucleus. THINK WHY!
Finally, the ethanol is used to solidify the DNA. In water, the DNA will go back to a liquid form, meaning DNA is soluble in water. When it is in ethanol, it uncoils and precipitates (becomes a solid) leaving behind the other cell components that are not soluble in ethanol.
Before you begin your experiment, you will need to write these down:
1. Create a problem statement in your journal. Perhaps the fundamental question we are asking is: What does DNA look like after it is extracted from _____ cells? Or, Does DNA extracted from _____ look the same as DNA from _____? 2.
2. Identify the manipulated and responding variables. What variable are you and your partners/classmates changing? What are you “measuring” as a result?
3. Set up a data section (charts/tables). What kinds of data are you collecting?
Pre lab questions: You must be able to answer these questions correctly before completing the lab.
1. Where in the cell is the DNA located? (Specifically)
2. What does the term DNA stand for?
3. What is the name of the model that Watson and Crick gave to describe the structure of DNA?
4. Why is DNA important? (What does it have in it that's important)
5. What molecule does DNA "code" for?
6. What chemical lysed (broke down) the membrane of the nucleus in your experiment?
7. What chemical solution (s) protected the DNA in order to be able to extract it from cells?
8. What chemical precipitates (transforms from liquid to solid) the DNA?
MATERIALS (Items with * are on your lab tray). Solutions will already be prepared.
*test tube rack *100 ml, 250 ml beakers
*4 corks or caps *Blender
*inoculating loop *graduated cylinders (10 ml and 100 ml)
*eye dropper/pipette *salt solution
*buffer solution *liquid detergent (soap) solution
*source of DNA *stirring rod
*4 test tubes *4 graduated containers w/ lids
*Microscope slide/cover
*Microscope Pipette-pump (for pipettes)
*test tube brush Mortar and pestle (if needed)
prepared buffer solution: 10% detergent solution
-57g granulated sugar -90 ml distilled water
-1 buffered aspirin (325 mg) -10ml Palmolive
-3 g Epsom salts
-add distilled water to 500 ml
*95% ice cold ethanol (poisonous if consumed!) Salt Solution:
-29.2 g. non-iodinated salt
-add distilled water to 250 ml
PROCEDURES for a basic DNA extraction (edit to fit your actual substance tested)
1. Get fashion wear-including goggles and aprons (if desired).
2. Cut off a section of your “living” substance (1 inch square) and place on a dish.
3. Combine 100 ml prep buffer and 10 ml detergent, and DNA source solution in blender. *Rinse graduated cylinders after each use.
4. Blend for 1 minute or until mixture is smooth.
5. Pour the mixture into a clean beaker and clean out blender thoroughly.
6. Using your graduated eye dropper/pipette, transfer 2ml of the liquid mixture, not the foam, to a test tube.
7. Add 2 ml of salt solution, screw on cork, and shake vigorously for 10 minutes.
8. After shaking for 10 minutes, look at mixture and note the layers:
--pellet (solids) will float and be in upper layer (looks like foam)
--lower layer is usually called the liquid or supernatant and has the DNA in it.
9. Carefully decant (pour off) the lower liquid layer into a clean test tube using a clean pipette. To do this, simply hold a glass stiring rod over the mouth of the tube. Make sure the solids stay in the first tube.
10. Use a 5ml pipette pump to slowly pour 5ml ice cold ethanol carefully down the side of the tube with the liquid, not the pellet, to form two layers of liquids. Mr. Heussman will assist with this step.
11. Let the mixture sit undisturbed for 2 minutes.
12. The DNA will float in the alcohol. The DNA should easily spool (collect).
13. Get a microscope, slide, and cover slip.
14. Use an inoculating loop to spool your DNA.
15. Take a small sample of the DNA and put it on the slide and place a cover-slip over slide. Observe under microscope. Draw, label and describe—follow microscope drawing protocol. This may take several trials or patience to “find” your DNA.
16. Clean up and have your teacher check your lab station. GET MY STAMP/INITIALS.
Reflection: Think about these as you write your discussion and conclusion.
10. What did you learn? What didn’t you find out?
11. What did you think was interesting?
12. How does this lab fit into what we've been learning in class?
13. Was this hard to do or easy? What would/could you change?