Materials Developed for Genetics and Molecular Biology

Team Member: Bud Bertino Cornell Institute for Regional Biology Mentors (CIRBM):
Curriculum Development and Instructional Strategies. Ithaca, NY, 14850
Copyright, August 1995
August 1995
CIRBM Developed Lab

DNA Extraction Lab

Teacher Section

• Overview
• Instructional Procedures, Part A: Teacher Directed
• Instructional Procedures, Part B: Student Design

Student Section

• Overview
• Part A: Introductory Problem
• Part B: Student Designed Experiment

Concept Map of Theme with Associated Labs

DNA Extraction Lab Teacher Section

Skills, attitudes and understandings:

• Demonstrate proficiency with the techniques involved in DNA extraction
• Extract DNA from various available sources
• Relate that plants, animals and bacteria are composed of cells which contain easily obtainable DNA
• Perform modifications in procedure (protocol) of basic DNA extraction and apply these modifications to the extraction of DNA from a variety of cells
• Devise a method of quantifying the yield of DNA and compare it with extractions from other DNA sources

Synopsis of the lab:

Students will extract DNA and test for its presence from unroasted (raw) wheat germ using a prescribed procedure. They will then attempt to modify the procedure to provide a less expensive, easier method. Students will also attempt to extract DNA from different available plant, animal and/or bacterial sources. The yield of DNA will be compared among the sources used.

Appropriate level biology course

This lab could be used with modifications from basic to AP biology

Length of the lab in minutes

• 45 minutes part A
• 3-4 additional 45 minute periods for part B

Materials

Part A:

For the teacher-led preliminary investigation, you will need the following for each team of 2-3 students in a class of students.

• 250 ml beaker or similar container (plastic beverage cup)
• hot plate
• non-roasted (raw) wheat germ
• thermometer
• pH meter or paper
• detergent - Palmolive or some other clear detergent
• test tube rack (beaker or something to hold a test tube at a 45° angle
• baking soda
• Adolph's natural meat tenderizer
• ice cold 95% ethanol (denatured ethyl alcohol)
• 15 ml (small) test tubes
• glass stirring rod, wooden skewer or eyedropper
• distilled water or tap water
• graduated cylinders (10 ml and 100 ml)
• DNA standard solution
• diphenylamine solution
• 4% salt solution

Part B:

These depend upon the student design but suggested materials for some design options are given.

• suggested materials to use as DNA sources include lima beans soaked in wat er for three days.(Use the bacteria growing in the smelly water surrounding the beans), liver, testes, onion and beef thymus.
• granulated sugar
• Epsom salts
• bufferin
• liquid detergent
• fresh papaya juice (substitute for meat tenderizer)
• pipettes
• test tubes & rack
• hot plate
• pH paper or meter
• thermometer
• ethanol
• Adolph's natural meat tenderizer
• non-iodized salt
• 1.5 ml Eppendorf centrifuge tubes
• mini centrifuge
• SDS (sodium dodecyl sulfate) 10% solution (replaces detergent)
• reusable coffee filter (used as strainer)
• DNA standard solution
• diphenylamine solution
• 4% salt solution

Directions for setting up the lab

Part A:

1. directions for preparation of materials and solutions, and culture of organisms:
   • Baking soda solution - dissolve baking soda in 500 ml water and check the pH. A pH of approximately 8 is desired. Continue to add baking soda until that pH is reached.
   • Set up glassware: Items on Part A list will accommodate student group s of 2-3 people.
2. No special preparation is necessary beyond the collection of materials.
3. general information on where/how to obtain materials:
   • All necessary materials are readily available except the raw wheat germ . This can be obtained inexpensively (approximately $ 1.00 per 12 oz bag from a health food store or co-op)
   • An additional step to the experimental procedure might be to add controls in the form of a test tube lacking wheat germ, another lacking detergent and a third lacking meat tenderizer to verify that no DNA will form if any of there items are missing from the procedure.
     
     

Part B:

This depends upon student design

• Have student teams decide how they will modify the original procedure. Provide all materials possible from the Part B list .
• Lima bean cultures can be established by placing 15 or so lima beans in a beaker and covering them with approximately five times their volume. Let culture develop for three to seven days or until a smelly scum of bacteria form. These huge bacilli are your source of procaryotic DNA.
• A 10% SDS solution can be purchased or prepared from powder.

Cooperative learning skills/strategies involved

Good working relationships and excellent record keeping are essential in the experience for reasonable results. A student should be designed to record all procedural modifications including amounts and/time changes used to improve the extractions. The group itself might split into subgroups to try different techniques for improvement of protocol (procedure).

• Student decisions and tasks include, therefore:
• lab design and method of data collection
• organization and rewriting of protocols

Background information for the teacher

DNA can be isolated from a variety of inexpensive, easily accessible sources. Plant cells (like onion) have been used successfully for this extraction. Animal cells like thymus and testes are also a rich source of DNA. Wheat germ has the unique qualities of being both an excellent source and extremely inexpensive.

Chemical indicator procedure for detecting the presence of isolated DNA includes the making of the following solutions:

1. Add 0.02 g DNA standard to 200 ml distilled water. Add one drop of glacial acetic acid. If required, heat gently to dissolve the DNA.

2. Dissolve 3.2 g diphenylamine in 200 ml of glacial acetic acid. Add 3 ml concentrated sulfuric acid. Store solution in a dark bottle. On the day of use, prepare a solution of fresh acetaldehyde (1 ml) in distilled water (500 ml). Add 1 ml of this solution per 100 ml diphenylamine solution.
3. Add 2 g of NaCl to 50 ml distilled water making a 4 % salt solution.
4. Hot plates with beakers of boiling water should be available to students when DNA testing is to be done.

Pedagogical information for the teacher

When DNA isolations are attempted, one of the following three outcomes result:

• No DNA results
• fluffy (sheared) DNA results
• DNA appears as long, continuous threads

Modification of techniques will hopefully produce DNA of the third type.

There are three basic steps in DNA extraction and isolation:

1. the cell must be broken (lysed) to release the DNA
   
   • detergent and salt solutions lyse the lipid cell membrane
2. the nuclear membrane (if present) must be ruptured
   
   • stirring breaks down cell walls, cell membranes, nuclear membranes
3. the extrtacted DNA must be protected from enzymes that will cause it to shear
   
   • sources tell us that the enzyme, DNAase, denatures at 60° and therefore if that temperature is maintained the DNA will not shear. DNA also may denature in the 60°- 80°C. A better procedure might be to keep the temperature lower and therefore reduce the DNAase activity.

Teaching tips

• This lab generates lots of enthusiasm and creative energy. Prepare yourself for being in great demand as a resource person.
• Keep the supply area well stocked but have your groups of student flow chart or concept map their new procedures before the OK is given by you to proceed.
• After each team has conducted their student design, it would be excellent to discuss all their results and describe their protocol modifications.. Each team could present their data to their classmates. This would give a broader understanding about the concepts to everyone and a pattern of techniques should surface.

Safety procedures:

• Work area should be uncluttered and clean.
• Safety glasses should be worn.
• Clean area thoroughly after completion of work.
• Return all supplies to appropriate location.
• Clean and return all glassware and materials as directed.

Instructional Procedures -- Part A: Teacher Directed

Introduction to the lab

Today great emphasis has been placed on DNA. Its importance lies in its ability to code information. This molecule is composed of four nucleotides which are themselves composed of alternating sugar (deoxyribose) and phosphate units and a nitrogenous base (either guanine, adenine, cytosine or thymine.

Questions for student interest or class discussion

• What does DNA look like?
• What is the significance of the sequence of nitrogenous bases in DNA? .
• Is DNA found in all cells?
• If DNA is to be removed from a cell what obstacles must be overcome?
• What parts of the cell would have to be altered or destroyed in order to extract the DNA?
• How do you test for DNA?

Hypothesis generation

Using the cookbook directions for DNA extraction, the class can discuss the purpose of each step in within procedure.

Data analysis and interpretation

• Students should carefully monitor each step in the procedure and report and changes in appearance of DNA solution noted.

Assessment items for this lab

Part A

• Amount and appearance of DNA isolated
• Overall cooperation within the group, division of responsibility
• Appropriate techniques

Part B

• Amount and appearance of DNA obtained
• Overall cooperation within the group, division of responsibility
• Lab procedure design

Rubrics for the assessment items

Part A:

• Amount and appearance of DNA isolated (1-2 points)
• Overall cooperation within the group, division of responsibility (1-2 points)
• Appropriate techniques (2-3 points)

Part B

• Amount and appearance of DNA obtained (1-2 points)
• Overall cooperation within the group, division of responsibility (1-2 points)
• Lab procedure design (2-3 points)

Suggested answers to data analysis and interpretation for student section:

1. DNA strands appear as long threads if unsheared or as fluffy pieces if cut.
2. DNA appears to be relatively insoluble in alcohol.
3. Value should be approximately .2-.3 gram/ 1gram of wheat germ used.

Suggested answers to questions/analysis for student section:

1. Heat helps accelerate the destruction of the cell wall, cell and nuclear membranes where present.
2. Stirring helps to break up the cell parts containing the DNA.
3. Iced alcohol helps to rapidly decrease the temperature and provide an interface across which the DNA will move. DNA is insoluble in alcohol.

Instructional Procedures --Part B: Student Design

Now that the students are familiar with the basic process of DNA extraction, they are ready to design an experiment to test some variables. They have already noted a procedure for DNA extraction from raw wheat germ. Now they need to design their own extraction procedure(s). Some points of discussion to lead the students into their design could include the following: These depend upon the student design but suggested materials for some design options are given.

• suggested materials to use as DNA sources include aqueous solution of lima beans (for bacteria cultured within the aqueous solution), liver, testes, onion and thymus.
• bufferin to alter pH during extraction
• a different liquid detergent to improve yield
• fresh papaya juice (substitute for meat tenderizer)
• varying the temperature
• 1.5 ml Eppendorf centrifuge tubes can be used to collect centrifugate, then dry it and weigh it
• mini centrifuge
• SDS (sodium dodecyl sulfate) 10% solution (replaces detergent)
• reusable coffee filter (used as strainer)

Once the class has discussed this topic, each team should select one variable to test and should design a lab to be implemented in class.

Potential hypotheses

• the yield of DNA per gram of original material might be increased by a change (improvement) in the procedure
• the yield of DNA per gram of original material might be improved if a different DNA source were used

Potential procedures

• Eliminate or alter the heating step. How does it effect yield of DNA?
• Choose a different DNA source. Does another saource of DNA produce a greater amount of DNA for each gram of material used?

Data analysis and interpretation

The raw wheat germ appears to be the best source of DNA for use in extraction. It is the least expensive, easiest to use ans seems to provide the greatest yield per gram. Although results may vary somewhat all of the items suggested as DNA sources should provide excellent yields.

References and suggested readings

• E.coli DNA Extraction by Rod Best, Sparta, North Carolina, published in NABT Biotechnology Sourcebook
• The Cookbook Translator by Anthony J. Bertino, Canandaigua Academy, Canandaigua, New York, and Lana Hays, Simon Kenton High School, Independence, Kentucky, published in Woodrow Wilson Biology Module 1993, A Further Look at Biotechnology
• Marmur. J. (1961). Journal of Molecular Biology, 3, 208-218.
• Teachers Guide to AP Biology Course, AP Development Committee fo the College Board, p. 63-65.
• The Onion Lab: DNA Spooling, Brown, J., Paint Brabch High School.

• DNA standard, Sigma, D-3159, 10 g ($12)
• Diphenylamine, Kodak Lab Chemicals #52, 101 9181, 100g, $32.10
• acetaldehyde, Kodak Lab Chemicals #52, 153 849, 250 g, $21.50

DNA Extraction Lab Student Section

Purpose of the lab

DNA stores genetic information which controls growth and homeostasis with all living cells and organisms. In this lab experience you will be asked to extract DNA from raw wheat germ.

Background information

All cells contain nucleic acids. Bacterial cells have no nucleus and therefore have their DNA unbounded by a nuclear membrane while plant and animal cells possess a nuclear membrane. Using detergents while stirring, you will attempt to break down the cell membrane, cell wall (plant and bacterial cells) and nuclear membrane, if present. Following this, the enzymes which might shred the long, thin fibers of DNA must be inactivated. If the procedure is done carefully, the DNA fibers will be released and can be seen and collected.

Safety notes

Safety goggles should be worn and the usual safe handling of materials and glassware observed. There are no caustic materials associated with this lab.

- - Part A: Introductory Problem - -

Hypothesis or prediction
__________________________________________________
__________________________________________________
Materials
Per class: 24 students working in groups of three.

• 8-250 ml beakers or similar containers (plastic beverage cups)
• 2-hot plates
• 25 grams non-roasted (raw) wheat germ
• 8-thermometers
• 8 vials pH paper or ph meter
• 1 bottle (12 oz) detergent - Palmolive or some other clear detergent
• 8 - test tube racks or beakers to hold a test tube at a 45° angle
• 1 box - baking soda
• 1 bottle - Adolph's natural meat tenderizer
• 100 ml ice cold 95% ethanol (denatured ethyl alcohol)
• 8 - 5 ml (small) test tubes
• glass stirring rod, wooden skewer or eyedropper
• distilled water or tap water
• graduated cylinders (10 ml and 100 ml)
• Per team: (of 2-3 students)
• For the teacher-led preliminary investigation, you will need the following for each team of 2-3 students
• in a class of students.
• 1- 250 ml beaker or similar container (plastic beverage cup)
• hot plate
• 1.5 grams non-roasted (raw) wheat germ
• thermometer
• pH meter or pH paper (range 5-9)
• 5 ml detergent - Palmolive or some other clear detergent
• test tube rack (beaker or something to hold a test tube at a 45° angle
• baking soda
• 3 grams Adolph's natural meat tenderizer
• 6 ml ice cold 95% ethanol (denatured ethyl alcohol)
• 2 - 15 ml (small) test tubes
• glass stirring rod, wooden skewer or eyedropper
• 100 ml - distilled water or tap water
• graduated cylinders (10 ml and 100 ml)
• 4% sodium chloride solution - 9 ml
• boiling water bath
• diphenylamine solution - 9 ml
• DNA standard solution - 3 ml

Procedure (Protocol)

1. Add 100 ml of water to a beaker and heat to 50°-60°C.
2. Add 1.5 grams wheat germ and mix until dissolved.
3. Add 5 ml detergent. Maintain 50°-60°C temperature andstir for 5 minutes.
4. Add 3 grams of meat tenderizer.
5. Make a baking soda solution of 50 ml water and a teaspoon of baking soda in a separate beaker. Use this solution to bring the pH of the wheat germ solution to approximately 8.
6. Maintain the 50°-60°C temperature and stir for another 10 minutes.
7. Remove from heat and place 6 ml of the wheat germ suspension in a clean test tube.
8. Pour 6 ml of ice cold ethanol carefully down the inside edge of the test tube so that it layer on top of the wheat germ suspension.
9. Let the mixture stand undisturbed for 5 minutes. Observe. DNA strands will appear at the interface between the ethanol-wheat germ suspension. Record your observations.
10. Weigh a small piece of filter paper (3X3 cm) and label in pencil with your initials. Record the weight. Using a pipette (eye dropper) draw up the DNA from the alcohol layer and place it on the filter paper square. Set aside and let dry overnight. Reweigh the next day. Calculate the DNA/gram wheat germ. Record.
11. Remove some of the DNA sample from the filter paper and place it in a labeled test tube containing 3 ml of the 4% salt solution and add 3 ml of the diphenylamine solution.
12. Into a second labeled test tube, place 3 ml of the 'standard DNA solution' and add 3 ml phenyamine.
13. To a third labeled test tube add 3 ml 4% salt solution and 3 ml phenylamine.
14. Place all three test tubes in a bioling water bath for 5 minutes and record the colors changes observed. Diphenyamine reacts with the deoxyribose of the DNA to produce a blue color, a positive indication of the presence of DNA.

Data analysis and interpretation

Answer the following questions:

1. 1. Describe the appearance of the strands of DNA? ______________________________________________________
2. 2. Make a statement about the solubility of DNA in ethanol.________________________________ __________________________________________________
3. 3. Calculate the yield of DNA from wheat germ.
   • To do this subtract the final dry weight of the filter paper + DNA ________grams from the filter paper dry (at start) ________grams ========= dry weight of DNA only -------------------------------------------------- ________grams
   • To calculate the yield of DNA per gram of wheat germ Divide the amount of dry weight DNA obtained by 1.5 grams of wheat germ. Yield = ____________/ gram wheat germ

Questions/Analysis

1. 1. What was the purpose of heating the wheat germ solution?______________________________ __________________________________________________
2. 2. Why was it so important to stir the wheat germ solution for 5 minutes?_____________________ __________________________________________________
3. 3. Why do you suppose that you used ice cold alcohol instead of room temperature alcohol?______ __________________________________________________ __________________________________________________

- - Part B: Student Designed Experiment - -

Now that you have competed the preliminary part of this lab, choose one of the following to complete with your group:

• design and test an experiment that can be carried out in the classroom for improving the extraction procedure of DNA from wheat germ without reducing the yield.
• use different sources of DNA and apply the original DNA procedure to determine if another source of DNA might produce a higher yield of DNA/gram of material used.

Hypothesis or prediction

From the information you now have regarding DNA extractions, develop a hypothesis that could be tested in a controlled experiment that will gather quantitative data.

Explain the reasoning behind your hypothesis.

Plan of Investigation

need to design a controlled experiment based on your hypothesis. You will need to make a numbered list of steps, similar to a recipe, that anyone could follow. You also need to consider the design for the table(s) or graph(s) that will be appropriate for recording your data. * Consider the following questions in designing your data tables and graphs: (You do not need to write out specific answers to these five questions)

1. What will you measure?
2. How will you measure your results?
3. What materials will you need?
4. How will you proceed with the investigation?
5. How will you show your results in data tables and graphs?

* Answer the following pre-lab questions. Submit the answers to your teacher before you start the lab.

1. What is the question you are investigating?
   __________________________________________________
   __________________________________________________
   __________________________________________________
   __________________________________________________
   
2. What variables are important?
   __________________________________________________
   __________________________________________________
   
3. What procedures would you use to test this?
   __________________________________________________
   __________________________________________________
   __________________________________________________
   __________________________________________________
   __________________________________________________
   __________________________________________________
   __________________________________________________
   __________________________________________________
   
4. What special materials will you need for this investigation, and where will you get them?
   __________________________________________________
   __________________________________________________
   __________________________________________________
   __________________________________________________
   
5. Describe the controls you have included.
   __________________________________________________
   __________________________________________________
   
6. What will you measure?
   __________________________________________________
   __________________________________________________
   __________________________________________________
   __________________________________________________
   
7. How will you graphically organize these data?
   __________________________________________________
   __________________________________________________
   __________________________________________________
   __________________________________________________
   

Lab write-up:

Each student should use the following format to write up Part B of this lab.You can work as a team to discuss the lab and analyze your results, but the written product should be done individually.

• Background
• Hypothesis
• Materials
• Procedure
• Results
• Discussion
• Conclusion

Additional Information: Include answers to the following questions as part ofyour lab write up. {Use any or all of the following as appropriate}

• How does your data relate to your hypothesis?
• What caused errors in your experiment?
• What other questions came from your results?
• To what other topics in bioology is this lab related? Include an explanation.
• What did your learn from this activity?

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