5. Label three 0.2 mL sterile PCR tubes [3 per student / 9 per group] of respective color with corresponding labels to the MM/PCR reactions you will prepare. Follow the table above. Keep tubes closed and work using aseptic technique to avoid contaminating the tubes. . Plant Codes = X, Y, or U . Student Code = 1- 12 . PCR amplicon = from A, B, or C primer sets . RED = A/Alul - B/BstNI restriction enzymes 3 tubes for each MM 6. Transfer 5 UL of each DNA sample into respective color /labeled tubes. Close the tubes & place on ice Use aseptic technique to avoid contaminating the samples. 7. Wait for the instructor to call your turn to go to the PCR station. Once you are at the PCR station: 8. Disinfect the aluminum foil by wiping it with 70% ethanol. 9. Verify that all the reagents are thawed. 10. Verify that all the samples are thawed. Spin the samples if needed to collect the contents at the bottom of the tube. 11. Place the reagents in order in the rack to ensure that you add them in order. 12. Wipe your gloves with 70% ethanol before starting to prepare the master mix. Assemble the PCR reactions on ice. Keep all tubes and pipette tips close at all times! 13. Obtain a sterile 0.5 mL tube and labeled MM and primer code (A, B, or C). 14. Assemble the Master Mix on ice as per table below [steps 1 to 4 on table]. a. Pipette each reagent carefully and slowly because the high content (50%) of glycerol in the storage buffer may lead to pipetting errors. Check the volume on pipette tip for accuracy. b. After transferring all the reagents, mix the reagents by pipetting up and down, close the tube and spin for 30 seconds to collect contents to the bottom of the tube. Volume Master Mix Vol (HL) / Order Component Final Conc. (PL) reactions* 1 Nuclease-free sterile dH20 N/A 2 2X Master Mix 1X 3 10 UM Primer Forward 0.5 UM 4 10 UM Primer Reverse 0.5 UM Total Volume of Master Mix [UL] 5 Template DNA 5.0 NA