Download Laboratory Manual - ILRI Research Computing

12. Perform PCR of samples in an ABI GeneAmp 9700 Thermocycler with heated lid (or equivalent PCR machine) using the following amplification program: Step Denaturation Denaturation Annealing Extension Final extension Soak Amplification Program Temp Time 94°C 3 min 94°C 30 sec 55°C 1 min 72°C 1 min 72°C 7 min o
20 C soak Cycles 1 30 1 1 13. At the end of the amplification programme put the PCR tubes on ice. 14. Label eight 0.5 mL Eppendorf tubes: 1 to 8, and add 3 µL 2X DNA Gel Loading Buffer to each tube. 15. After the PCR has completed, add 3 µL of PCR products to the tubes containing 2X DNA Gel Loading Buffer. Use a clean pipette tip for each PCR product. 16. Run on a 1.8% agarose gel with GelRed. Run at 50-­‐100 V in 0.5X TBE buffer until the Bromophenol Blue dye has run 2/3 the length of the gel or the Orange G dye has run 3/4 the length of the gel. 17. Document the gel with the gel doc system. 18. A predicted rbcL PCR product for Zea mays is 634 bp (using primers with M13 tails). Sizes of rbcL PCR products from other plant species may vary slightly. 19. Store the remaining PCR products at -­‐20oC. Example: rbcL PCR with titrated MgCl2. 1.8% agarose gel. 69