PCR Protocol - Part 2

Summary

This segment explains how to add DNA to prepared PCR reactions and run them in a thermocycler. It emphasizes careful labeling, thorough mixing, collecting liquid at the bottom of the tubes, and confirming that the selected program starts correctly.

Procedure and Key Instructions

1. Divide the PCR Cocktail

• Split the collected cocktail solution among the five PCR tubes.
• PCR tubes are supplied in strips of four, so five reactions require two strips.
• Add 24 µL of cocktail solution to each tube.

2. Add DNA Samples

• Label the tubes before adding DNA.
• Mix each DNA sample before transferring it.
• Add 1 µL of DNA to each reaction. Although this is a very small volume, a drop should be visible in the pipette tip.
• Place the tip at the bottom of the tube. Pump the pipette plunger up and down, then press to the second stop to help expel the full volume.

3. Mix and Collect the Reaction

• Cap the tubes tightly and flick them to mix.
• Use a mini microcentrifuge with an eight-tube strip head to bring the solution to the bottom of the tubes.
• Mix and collect solutions before each step, especially if they have been sitting.

4. Run the Thermocycler

1. Turn on the thermocycler using the switch on its back.
2. Press the metal latch and lift the lid to insert the tubes. They can go in any position or orientation.
3. Close the lid and confirm that the latch clicks into place.
4. Use the horizontal arrows to navigate to the preprogrammed PCR program for the target. The subtitle identifies the example as “Actn 10 and three,” apparently referring to ACTN3.
5. Press Proceed to start the program.
6. Enable the heated lid when prompted. This helps prevent condensation at the top of the tubes.
7. Confirm that the instrument reaches the initial temperature and begins timing without errors. Once the run has started properly, it can proceed unattended for several hours.
8. When the run is complete, turn off the thermocycler using the rear switch.

Speakers and Sources

• Speaker: One instructional narrator; no individual is named.
• Video source: The Jackson Laboratory.
• Other audio: Background music.