DNA Extraction of Your Samples for PCR 1 Prepare three
Last updated: 8/27/2023
DNA Extraction of Your Samples for PCR 1 Prepare three samples Your food sample the GMO positive sample and the GMO negative sample Reducing the chance of contamination is critical Wipe all tools used micropestle and razor blade with alcohol wipes and do not switch tools between samples Crush or dice up a small amount of your sample with a micropestle or razor blade Add the crushed sample to a clean 1 5 mL Eppendorf tube to a level about halfway to the 0 1 mL mark Label the tube with the sample type and your group color Repeat the process for the GMO negative and GMO positive sample 2 Add 100 L of Edward s buffer to each tube containing the plant or food material Remember to change tips between samples to decrease the chance of contamination 3 Twist a clean micropestle against the inner surface of the 1 5 mL Eppendorf tube to forcefully grind the plant tissue or food product for 1 min 4 Add an additional 900 L of Edward s buffer to each tube containing the ground sample Grind briefly to remove tissue from the micropestle Vortex the tubes for 5 sec 5 6 Boil the samples for 5 min in a water bath using a circular floater Wrap tube openings with parafilm or use lid locks to keep lids from popping open during heating 7 Place the tubes in a balanced microcentrifuge and spin for 2 min at 12 000 rpms to pellet cell and food debris 8 Transfer 350 L of each supernatant to a fresh tube Maintain labels for each sample type and group color Be careful not to disturb the pelleted debris when transferring the supernatant Discard old tubes containing the precipitates 9 Add 400 L of isopropanol to each tube of supernatant 10 Mix by inverting the tubes several times and leave at room temperature for 3 min 11 Place the tubes in a balanced microcentrifuge and spin for 5 min at 14 000 rpms Align tubes in the rotor with the cap hinges pointing outward Nucleic acids will collect on the tube side under the hinge and pellet out of solution during centrifugation 12 Carefully pour off the supernatant from each tube and then completely remove the remaining liquid with a micropipette set at 100 uL