Procedure Page

The purpose of the Polymerase Chain Reaction is to amplify a desired section of DNA. The multitude of copies will be double-stranded, just like the original. PCR requires two primers that work from opposite ends of the targeted segment of DNA. For an excellent animation of the process, as it happens in the thermalcycler, see Cold Spring Harbor Labs.

1. Bring DNA extractions to room temperature.

2. Label PCR tubes to correspond to DNA samples. Also label one tube as a negative control. The tubes hold a volume up to 200 μl. The bead contains buffer, dNTPs, and Taq Polymerase in a solid form that is stable at room temperature.

[If beads are not available, you can use an alternative recipe for the components.]

3. Add 1 μl of DNA to the PCR tube. Avoid touching the bead in the bottom of the tube. Do not add any DNA to the negative control.

4. Add 24 μl of prepared PCR primer cocktail* to the PCR tube.

5. Make cocktail according to the number of samples you have.

6. PCR primer cocktail recipe:

1 μl of primer

2 μl of Big Dye (contains dNTPs,    ddNTPs, enzymes)

                                        6 μl sterile distilled water

7. Place tubes in the thermalcycler. Set conditions according to primer annealing requirements.

8. Run 7 μl of the results (with 1 μl loading dye) on a 2% agarose gel to check the results.