Does your food contain GMO DNA? DNA Extraction 1. Place approximately 1-2 mm3 of your food sample to a clean 1.5 ml tube. 2. Macerate tissue using a blue pestle and hand homogenizer for 30-60 seconds. You will still have some solid pieces, but you should have a slurry. If your tissue is very dry such as tortilla, you may need to add 50 µL of extraction buffer to aide in homogenization. 3. Add 400 µL (or 350 µL if you have already added 50 µL) of extraction buffer (200mM Tris HCL at pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS). 4. Vortex for 5 seconds. 5. Centrifuge the extract at 13,000 rpm for 3 minute. 6. While centrifuging, add 300µL of isopropyl alcohol to a fresh Eppendorf tube. 7. Add 300µL of the supernatant into the isopropyl alcohol tube, take care to not bring any solid material, you may need to add less than 300µL. 8. Leave the mixture at room temperature for 2 minutes. 9. Centrifuge at 13,000 rpm for 5 minutes. 10. Quickly pour off the liquid, making sure not to dislodge the pellet. 11. Close tube, centrifuge for 10 sec. 12. Remove remaining liquid with eppendorf pipet. 13. Add 1 mL of 70%-75% ethanol to the Eppendorf tube. 14. Centrifuge at 13,000 rpm for 2 minutes. 15. Quickly pour off the ethanol making sure not to dislodge the pellet. 16. Close tube, centrifuge for 10 sec. 18. Remove remaining liquid with eppendorf pipet, and leave the Eppendorf tube open for 15-20 minutes at 37°C. Your sample should be dry and should not smell of ethanol. 18. Add 100µL sterile water to the Eppendorf tube to dissolve the pellet. You may need to pipette the liquid up and down vigourously several times to resuspend the pellet. It is highly likely that much of the pellet will not dissolve. 19. Centrifuge at 13,000 rpm for 3 minutes to pellet the debris that did not dissolve in TE and pipette the liquid into a clean sterile tube. The DNA should be dissolved in this water and can now be used for PCR amplification. Be sure your tube is labeled. PCR Procedure 1. Mix each of the following into a PCR tube: a. 10 µL of GoTaq b. 4.5 µL of each of two primers (forward and reverse) c. 1 µL DNA extract 2. Mix the mixture by tapping on the PCR tube. Your total volume will be 20 µL. 3. Centrifuge briefly to pool the PCR ingredients if necessary. 4. Place the prepared PCR tube into the PCR machine and run using the following sequence: a. Heat to 95º C for 5 minutes b. Cool to 95º C for 30 seconds c. Cool to 57º C for 45 seconds d. Warm to 72º C for 30 seconds e. Repeat this sequence 35 times beginning at step b f. Hold at 72º C for 7 minutes before taking to 4º C for storage g. Samples are now ready for gel electrophoresis Gel Electrophoresis 1. Load 10 µL of molecular weight marker into one of the electrophoresis gel wells. 2. Load 10 µL of the sample into another well of the electrophoresis gel. 3. Repeat step 2 (using sequential empty wells) until all PCR samples have been loaded into the gel. 4. Run the electrophoresis between 100 and 125 V. 5. Stain gel with Ethidium Bromide. PCR worksheet Reagent name One Check if reaction added (µL) GoTaq 10 F-primer 4.5 R-primer 4.5 DNA extract 1 Total volume 20 2X GoTaq (contains Taq polymerase, dNTPs, MgCl2, and buffer for ideal reaction and loading dye for electrophoresis) Project Name: _____________________________ Researcher initials:______ Date:____ Samples loaded in gel: well 1 sample 2 3 4 5 6 7 8