General Protocol for Isolation of DNA

DNA Extraction : Low Tech You have probably seen many methods of extracting DNA as you search the Web and explore your teacher materials. The process gets simpler as time goes by . In the lab we saw many methods of DNA extraction demonstrated. You may pick out your favorite and use it until another comes your way. Remember the main components of the reaction: Lysing the cell membranes with detergent or SDS Releasing the DNA from the histones, setting up an ionic environment Floating the DNA in alcohol, density gradient And Spooling the DNA to see the product and test purity of product. Consider exploring one or more of the variables demonstrated in these lab presentations with DNA extractions. Enjoy.

LAB 2 Protocol 1: Food DNA Extraction Protocol adapted from http://www.hhmi.princeton.edu/hhmi/html/manual.html

1. Materials and Equipment (per lab group): a. three 30g samples of fresh banana b. Preparation of solutions of detergent, alcohol, and protease is described below. c. fresh pineapple juice (bromelin enzyme) or d. meat tenderizer (papain enzyme) e. mortar and pestle f. four 5 ml pipets and bulbs g. Hot water bath at 65o C--the usual temperature of very hot tap water h. four 6” squares of cheesecloth—4 single layers each i. four 50 ml centrifuge plastic tubes j. Centrifuge k. six 15 ml plastic test tubes (or culture tubes) l. optional--1.5 ml plastic tubes (for storing DNA) m. Pasteur pipets with ends flamed and bent into small hooks for spooling DNA n. plastic spoon narrow enough to fit into 50 ml tubes o. Ice and ice bucket p. plastic funnel q. Gloves 2. Preparation of Solutions (for 3 trials for one lab group): a. Detergent: 3 ml 50% Palmolive or Wisk Free liquid (1.5 ml water + 1.5 ml detergent) • •

b. Protease (either meat tenderizer or pineapple juice): Meat tenderizer: add 4 g of Adolf’s meat tenderizer to 10 ml of D.I. water, mix Pineapple juice: cut up fresh pineapple and blend on high to puree, strain through 4 layers of cheese cloth, collect at least 3 ml c. Alcohol: 10 ml ice-cold or freezer-cold 95% ethanol d. Sterile deionized or distilled water

3. Isolation Procedure for each trial: a. Wearing gloves, each group should put 10 g banana and 1 ml cold water into a mortar and pestle and grind the banana until they appear smooth and creamy. b. Add 1 ml of 50% detergent and 1 ml of protease source. c. Use a spoon to scrape the banana mush into a 15 ml plastic tube and incubate

in a 65o C hot water bath for 10 minutes to lyse the cells. Transfer the tube containing the mush to ice to stop the lysis. d. Place four layers of cheesecloth into a funnel and put the neck of the funnel into a centrifuge tube. To filter out large chunks of debris, transfer the mush into the cheesecloth and use your (gloved!) fingers to twist the cheesecloth, thus squeezing liquid into the tube. There should be about 5 ml, but the amount is not important. e. Spin the tube in a centrifuge at about 3,000 rpm for five minutes f. Pour the supernatant produced into a clean 15 ml plastic tube. Slowly pipet or pour an equal volume of 95 % ethanol that is ice-cold or at freezer temperature (-20o C) carefully down the side of the tube, forming two phases. The alcohol should be added slowly to avoiding mixing with the aqueous phase. The upper phase is the alcohol, and the bottom aqueous phase contains the DNA. g. Let sit on ice undisturbed for at least 5 minutes or more if possible. After about 5 min, you should see air bubbles and cream-colored fibers of DNA slowly accumulating at the interface. If the yield of DNA is really high, after longer times the DNA fibers may form a mat that rises into the ethanol--a carpet of DNA-definitely cool! h. If DNA is visible at the interface, hook it with the hooked Pasteur pipet and drag it along the side of the tube to transfer it to a 1.5 ml microtube filled with ethanol. Carefully put the DNA into 1.5 ml tubes by scraping it off the glass rod against the lip of the tubes or using a second hook to scrape it off. DNA can be saved for later analysis such as drying out & weighing or staining with methylene blue. i. Repeat isolation steps 2 more times to verify results

Analysis Questions: 1. • • • • • •

What is the purpose of the following in DNA extraction: DetergentProteaseEthanolHeatingFiltering with cheeseclothCentrifuging-

2. Should you be able to see a double helix structure in your extraction? 3. What type of tests would you do to verify that you have DNA?

4. 5. Write an experimental design testing one variable that affects the DNA yield. Include methods of how you would compare yields.

Teacher Notes • •

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In addition to the banana, other fruits or vegetables such as kiwi, strawberry, spinach or others can be used. In one trial, we used multipurpose contact solution as a possible protease source, yet it does not contain true proteases. Rather, it contains detergents, EDTA, hydranates, and citrates that act as chelating agents. EDTA sequesters metal ions which are cofactors in many DNAases and other proteins which degrade DNA. Hydranate attaches to proteins and separates them by repulsive ionic forces. Together these additives separate proteins from the DNA and protect DNA from fragmenting. When we tried liver, the homogenate congealed to a semi-solid in the hot water bath and therefore was not usable. If meats are used as the DNA source, other mechanisms for lysing cells should be used. Bacteria was also used as a source of DNA, but very low yields were detected most likely due to the low number of cells in suspension. If trying this again, we would use 10ml of a highly concentrated bacterial suspension. What is essential in this experiment is to make sure that the cells are completely lysed, by mechanical means as well as detergents. Also, proteases and chelating agents (EDTA, hydranates, citrates, etc) are necessary to increase the DNA yield; however, we were able to extract minimal amounts of DNA without a protease source. After initial spooling of DNA mat, place parafilm over culture tube, invert and shake to mix the alcohol with DNA at the bottom of the liquid phase. This will precipitate more DNA. Let tube sit for 5 minutes, then spool again. Precision and accuracy in measurement of the additives seemed to be irrelevant to DNA yield as long as there is consistency in all three trials. Try to avoid as much cell debris in each filtering stage to ensure a cleaner, more readily visible extraction.

Protocol 2: Kitchen DNA Extraction (Low Tech) Plant Prep: Materials: Split green peas soaked overnight in warm water Dawn dishwashing liquid Salt 70% Isopropyl alcohol, or 95% Ethanol 250 ml beaker kitchen strainer mortar and pestel or small food processor Methods: Crush or grind the peas and add about 50 ml water to the mixture. Strain the peas through the kitchen strainer conserving the liquid. To the liquid, add a generous squirt of Dawn liquid and swirl the beaker for about two minutes being sure not to make bubbles. Add a pinch of salt and swirl for two more minutes. Let sit about 5 minutes. Layer alcohol on top of the liquid by pouring the alcohol down the side of the beaker being sure not to mix the liquids. Watch the DNA bubble up in the alcohol. Using a clean glass rod, insert the rod into the beaker and twist gently The DNA will spool on to the glass rod. Animal Tissue Prep: Materials: Substitute animal organ meats for the plant materials, use a small amount (1 gram or so) Substitute Adolph's Meat Tenderizer (unseasoned) for the salt. Method: Same as above.

Protocol 3: Wheat germ extraction 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

100 ml H2O, heat to 50-60 C +1.5g specimen, mix until dissolved +5 ml detergent (Wegmans dish detergent) keep @ 50-60C, stir for 5 min. +3g Adolph’s tenderizer (0, 3g, 6g) + baking soda to pH 8 keep @ 50-60 C, stir for 10 min. remove from heat + 6 ml solution to test tube, cool to room temp. + 6 ml iced cold EtOH (50%, 75%, 100%), pour down side of beaker sit 2-3 min use glass rod to spool DNA pipette DNA off onto pre-weighed notebook paper dry for 1 hour weigh paper with dried DNA

Protocol 4: The Extraction and Isolation of DNA Examining the Effects of Various Alcohols

Preparation: 1. Premix an extraction buffer by combining the following: • 4.4 g sodium chloride (0.15 M) • 16.05 g sodium citrate (0.15 M) • 50 ml of dishwashing detergent • Dissolve with distilled water to 500 ml. 2. Obtain 25 g of each of the following (or a suitable replacement) to represent four of the six kingdoms: • B. subtilis (bacteria) • Mushrooms (fungus) • Banana (plant) • Liver (animal) Group note: This 25 g sample is enough for a class working in large groups: it will yield about 6 samples. There will be plenty of extraction buffer for at least 2 classes. 3. Set up 2 water baths, one hot and one cold. The hot water bath should be approximately 60 degrees Celsius, the cold water bath should be prepared with ice. 4. Obtain a variety of alcohols to test (i.e. vodka, 70% isopropyl alcohol, 95% ethanol) in order to determine which is the more effective. Instructions: (to be used for all of the samples) 1. Prepare samples of the bacteria, plant, fungus, and animal samples. Samples should first be crushed, and then blended in the blender with distilled water. 2. Choose which sample kingdom to test first. Transfer 25 g of the desired sample to test tubes #1, #2, and #3. Add 20 ml of extraction buffer to each of these test tubes 3. Place the three test tubes in a 60 degree Celsius hot water bath for 15 minutes. 4. Remove the three test tubes from the hot water bath and place them into the ice-water bath for 10 minutes. 5. Add 5 ml of the chilled isopropyl alcohol to test tube #1, 5 ml of chilled vodka to test tube #2, and 5 ml of chilled 95% ethanol to test tube #3. Pour this down the side of the test tube. Invert the test tube and/or tap at the bottom of the tube with one finger in order to mix the contents. 6. Set aside test tubes for DNA to precipitate out of the chosen alcohol.

7. Using a stirring rod (and some source recommend wood over glass), spool the DNA out by twirling the rod gently around the interface.

Observations and Results:

Variables Isopropyl alcohol Vodka 95% ethanol alcohol

Liver

Banana

B. subtilis

Mushrooms

Rubric for DNA Isolation: • 3 – DNA was visible in the test tube as a precipitate. When the DNA was spooled, a visible sample was produced. • 2 - DNA was visible in the test tube as a precipitate, but spooling did not provide a visible sample. • 1 – There seems to be traces of DNA visible in the sample (murky, white precipitates floating throughout, etc.) • 0 – There is no DNA visible. Recommendations: • This protocol is not appropriate for the liver sample, as written. It was extremely concentrated. Either a different protocol should be used or meat tenderizer added to the extraction buffer. • The temperature of the alcohol could also be varied to determine if there is an optimum range within which DNA precipitates.

Assessment Questions: • This protocol was originally intended for an onion cell. Based on this, which sample should yield the best results? Explain your reasoning. •

Based on the results from the data table, does there seem to be an alcohol that is more effective than the others? Or, does it vary by kingdom? Explain.

•

Which sample would be affected the most if meat tenderizer was added to the extraction buffer recipe? Explain your answer.

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Document the major steps of the DNA Extraction and Isolation method. Explain the importance – and function – of each step.

•

Describe how the final DNA would be affected if the following steps were altered: o the detergent was left out of the extraction buffer o the hot water bath reached 100 degrees Celsius when the samples were in it o the samples were only cut into small pieces versus being macerated in the blender

Our Results: Variables Isopropyl alcohol Vodka 95% ethanol alcohol

Liver 1

Banana 2

B. subtilis 3

Mushrooms 2

2 1

2 3

2 3

2 2

Protocol 5: Assessing the efficiency of extraction Materials: Organisms Banana Salad mushroom Liver Bacillus Subtilis

Water NaCl liquid detergent meat tenderizer isopropyl alcohol

250ml beaker test tubes (1 per student) sieve filter paper glass stirring rod

DNA Extraction Protocol 1. Place in a blender jar: • 42 grams (50 ml or 1/4 cup) of Banana or any other DNA source • 100 ml (1/2 cup) of water • A pinch of salt (1/2 gram or 1/16 teaspoon). 2. Blend on high for 15 seconds. 3. Strain the mixture through a sieve for 5 minutes to remove the unblended particles. 4. Place the filtrate in an ice bath cooled to 1° C. 5. Add 10 ml (1 tablespoon) of detergent. Swirl to mix. Let sit for 5 – 10 minutes. 6. Add a pinch of meat tenderizer 7. Fill a test tube about 1/3 full with the liquid 8. Tilt the test tube and slowly pour an equal amount of alcohol down the side of the tube so that it layers on top of the split pea liquid. 9. Stringy DNA should appear at the boundary between the filtrate and the alcohol. 10. Use a wooden stick or a hook to gently move the split pea liquid up into the alcohol so that more DNA will precipitate out; you can also let the tube sit for 30 minutes or more. 11. You can keep the DNA indefinitely in sealed container with alcohol or dry it on paper. 12. Repeat steps 1-11, altering step 4 to keep the filtrate at room temperature. 13. Repeat steps 1-11, altering step 4 to keep the filtrate heated to 55° C. 14. Record results. 15. Repeat steps 1-14 using mushroom, liver, and Bacillus subtilis Protocols available at the Genetic Science Learning Center • Green Pea: http://gslc.genetics.utah.edu/basic/howto/teacher.html • Student handout for protocol: http://gslc.genetics.utah.edu/basic/howto/handout.pdf

Results: Organism Banana Banana

Temperature 1° C 18° C

Banana Liver

55° C 1° C

Liver

18° C

Liver

55° C

Mushroom Mushroom

1° C 18° C

Mushroom

55° C

B. subtilis B subtilis B. subtilis

1° C 18° C 55° C

DNA Yield No results DNA visible, but quantity was not enough to spool No results Good results; spoolable; .00 g DNA visible; nonspoolable DNA visible; spoolable;.07g No results Fuzzy DNA; nonspoolable Pure visible DNA, spoolable, .002g No results No results Pure visible DNA, nonspoolable

Summary: Banana produced the greatest yield at room temperature, while the bacteria, mushroom, and liver all produced the greatest yield after being heated to 55° C. Possible Sources of Error: • Concentrations: o Amount of filtrate collected affects detergent concentration o Meat Tenderizer: role relative to organism • Contamination: o Blender, stirring rod, non-sterile environment • Temperature fluctuation: o Fluctuation of ice bath & hot water baths • Time o Timing between filtration and addition of detergent varied greatly o Timing between addition of detergent and addition of alcohol • Spooling o May have received greater yields using pasteur pipette o Ultimately received no quantifiable data • Addition of Alcohol o It is critical that the alcohol does not mix with the filtrate, but forms a layer on top of the filtrate. If the alcohol is misapplied, the yield can be adversely affected.

Evaluation of Protocol • Provided visible, but non-quantifiable results • Small amount of protease added at the end of the procedure may have contributed to the unspoolable DNA, even with large visible yields, due to uninhibited enzyme activity throughout the extraction • Procedure was most effective with liver, though the DNA was clearly contaminated with other molecules. • Effective as a crude extraction using cheap and readily available materials. Conclusion Using the protocol provided by the genetic science learning center, most organisms provided the greatest DNA yield when the extraction occurred at 55°C. This result is contrary to our original hypothesis that cooling was necessary to produce the greatest yield. Our belief is that the heating may promote additional cell lysis, releasing more DNA into the filtrate than in the other methods. The heating may also promote the action of the protease, enabling more DNA to be released from the histones. Extensions Several follow-up investigations would be useful, including the study of this protocol on organisms such as the onion that traditionally produce large DNA yields in crude extractions. The effectiveness of this protocol is questionable, especially in light of the small amount of protease used and the application of this protease towards the end of the extraction. It would be optimal to test the heating technique on a protocol known to provide spoolable quantities of DNA, such as the protocol used at Pine Crest School.

Protocol 6: DNA Extraction-The “Banana Daiquiri” Method Purpose: To develop a protocol for teaching DNA extraction, using one procedural variable and bananas. Materials*: Blender Ice Bath 95% Ethanol 4 x 250 mL beakers Meat tenderizer Coffee filters Labeling tape Glass stir rods White paper *All bold-face materials could be varied in student protocols.

15% NaCl solution 2 x 15 mL culture tubes Banana

Procedure: 1.)Place ½ of banana into the blender. Cover with 100 mL of 15 % NaCl solution. 2.)Add 25 mL of detergent. (variable) 3.)Blend into smithereens!! 4.)Strain through 3-4 layers of cheesecloth into 250 mL beaker. Divide into smaller test tubes (approx. 6 mL in each tube) 5.)Add pinch of meat tenderizer (variable) 6.)Cut pieces of white paper into small squares (approx. 3 cm sides). Mass these squares and mark them with different variables marked. Put mass of paper on side. 7.)Add 6 mL of ice cold 95% ethanol. Layer on top of sample mixture. 8.)Stir the area between the layers with a glass rod or wooden splint. (DNA should stick to stirring rod). 9.)Transfer all the DNA you can extract from each sample to the white paper. Place these pieces of paper into the hood to dry overnight. 10.)Next morning, weigh the individual pieces of paper. Calculate the weight of DNA (subtract new weight from weight of paper alone). Teaching protocol: • Lab (group) assessment 1. Appropriate lab behavior displayed (incl. safety, use of equipment) 2. Following of procedures-creation of extracts 3. Successful spooling of DNA • Written (individual or group) assessment 1. Data table of DNA yield (grams) 2. Sample analysis questions • What purpose did the alcohol serve in the extraction? • What purpose did the salt serve in the extraction? • What purpose did the meat tenderizer serve in the extraction? • Discuss the physical properties of the DNA molecule which allow the molecule to be extracted. 3. Complete lab report • Alternative exercises which may include: 1. Different organisms (our procedures worked with liver) 2. Testing other variables (salt, temperature, etc.)