DNA Extraction Lab Do all cells contain DNA? Do certain organisms contain more DNA than others? The goal of this DNA extraction lab is to answer these questions as a class. Please read the DNA extraction protocol below and answer all the pre-lab questions. Come prepared on Thursday, August 14th, to run this lab with a partner. In addition, if you have a food item that you would like to test for DNA, feel free to bring it in as well (if you plan to do this, please email me to make sure your item is ok and will yield DNA). 1. Write the samples that you plan to use: Positive Control: ______________________ This is a sample that is known to contain DNA (we will use banana). Negative Control: _____________________ This is a sample that is know to NOT contain DNA (what would YOU use?) Experimental: ____________________ This is the sample you will try and determine the amount of DNA it contains… if it contains any! Materials: 1 capped empty test tube for positive control (“+“) 1 capped empty test tube for negative control (“-“) 1 capped empty test tube for experimental sample (“E”) 3 labeled tubes filled with the following: DNA Buffer (Composed of 5 ml dishwashing liquid + 1.5 g NaCl + 5 g NaHCO3 + 120 ml distilled H2O) Distilled Water Ethanol (on ice) 5 plastic labeled pipettes (“DNA Buffer, Ethanol, E, PC, and dH 2O) Experimental sample (liver, lettuce, raw wheat germ…) Control sample (banana) 1 mortar and pestle #2 filter or cheesecloth 1 small beaker 1 glass stirring rod 1 test tube rack 2 spatulas or spoons + plastic weighing boat Electronic balance DNA Extraction Procedure: 1. Go to your lab station and make sure all materials above are present. 2. Using a spatula or spoon, weigh out 7.5 g each of your positive control and experimental samples. 3. Place your positive control sample (banana) into the mortar. Using the plastic pipettes labeled “dH2O” and “DNA Buffer,” add 7 ml of distilled water and 3 ml of DNA buffer to the mortar. Using the pestle, gently mash up the sample and mix well with the distilled water and DNA buffer. 4. Carefully pour off all of the liquid experimental mixture (avoid solid chunks) into a beaker or cup. You may need to use cheesecloth or a #2 filter here…you want a translucent liquid, not lots of solid matter mixed in. 5. Use the “PC” plastic pipette to add 2 ml of this liquid sample into the “+” tube. 6. Wipe out and rinse the mortar and pestle. Repeat steps 3-5, but this time for your experimental (“E”) sample. 7. Now transfer 2 ml of distilled water into the negative control tube, again using the dH2O pipette. 8. Add 1 ml of DNA buffer (again using the “DNA Buffer pipette) to each of the three capped test tubes. Be careful not to touch the tip of the pipette to the inside of the test tubes or your samples may become contaminated. Re-cap the tubes and mix well by inverting (not too much; we don’t want too many bubbles). Allow the tubes to sit in the rack for at least 5 minutes (the longer you wait, the better your results). 9. Add 2 ml of ethanol (using the “Ethanol” pipette) slowly down the side of each of the three tubes to form a layer that floats on top of each sample. DO NOT MIX THE SAMPLE. If DNA is present it should precipitate out in white or clear clumps that may also look like cobwebs or gooey strands. Again, the longer you wait here, the better your results. 10. How will we quantify our results? _____________________________________ _________________________________________________________________ _________________________________________________________________ _________________________________________________________________ Pre-Lab Questions: Your answers will be collected on the first day of class. Be prepared to discuss your answers in class on Thursday, August 14 th. 1. Write two hypotheses that address each question (If…then…because format; don’t forget to address each of your controls AND your experimental) a. Do all cells contain DNA? b. Do certain organisms contain more DNA than others? 2. Refer to the contents of the DNA Buffer in the materials section. What is the function of each of the components of this mixture? Support your answer with detail. (Hint: Hunt around on the Internet if you’re lost). 3. Begin to think about how you will prepare a data table to collect the class data, as well as your group data. We won’t know exactly how this data table will look until we know which groups are testing which samples.
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