Amplification (PCR)

Amplify DNA fragments through Polymerase Chain Reaction

When a gene (not the entire chromosome) is in a low abundance, this quantity of the gene can be amplified in an exponential manner. Taq polymeraise, a biological machine (often in the form of PCR beads) is added to the DNA sample. The tube with DNA solution is placed into a PCR machine, like the Revolutionary Science RevCycler. During the 94 Celsius denature phase, the DNA ladder denatures (separates into two half ladders) and the temperature of of the PCR machine drops to the anneal temperature (usually between the temperature of 40 to 60 Celsius), so the RNA primers can attach to the gene of interest. The next step is extend at 72 Celsius, where the RNA primers are removed and replaced with single stranded DNA, to complete two new DNA ladders. This process is repeated. Each cycle doubles the original amount of DNA. This usually continues for about 35 cycles. When the DNA is amplified it can be left in the RevCycler where it will automatically change temperature to a 4 degree Celsius refrigeration temperature hold. Amplified DNA can be stored in a freezer until analysis. Analysis can be performed with DNA electrophoresis chamber like the DNA RevBox.

  1. Obtain PCR tube containing PCR beads. Label the tube with your identification number.’
  2. Use a micropipet with a fresh tip to add 22.5 uL of one of the following primer/loading dye mixes to the tube: plant rbcL, fish Col, or insect-mammal Col. Allow the bead to dissolve for 1 minute.
  3. Use a micropipet with a fresh tip to add 2.5uL of your DNA (from Part I) directly into the appropriate primer/loading dye mix. Ensure that no DNA remains in the tip after pipetting.
  4. Store your sample on ice until you are ready to begin thermal cycling.
  5. Place your PCR tube, along with those of the other students, in a thermal cycler that has been programmed for 35 cycles of the following profile:
    • Denaturing step: 94C 30 seconds
    • Annealing step: 54C 45 seconds
    • Extending step: 72C 45 seconds

The profile may be linked to a 4C hold program after the 35 cycles have been completed

After thermal cycling, store the amplified DNA on ice or at -20C until you are ready to continue with Part IV.

 

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