PCR Protocol - Part 1

Main Ideas and Procedure

The video explains how to calculate and prepare a PCR master cocktail for a group of samples. Combining reagents shared by all reactions reduces repeated pipetting and the chance of errors.

1. Complete the Worksheet and Calculate the Cocktail

• Record the samples and primers to be used on the worksheet.
• Calculate the number of PCR tubes required, including a negative control.
• Add one extra reaction to allow for pipetting loss or error.
• Multiply each reagent’s per-reaction volume by the total number of reactions to determine how much to add to the cocktail.

Example: Four DNA samples plus one negative control require five PCR reactions. With one extra reaction, calculate the cocktail for six reactions.

Reagent Volume per reaction Volume for six reactions RedTaq Ready Mix 12.5 µL 75 µL Molecular biology water 11.5 µL 69 µL Forward primer 0.5 µL 3 µL Reverse primer 0.5 µL 3 µL Total 25 µL 150 µL

2. Label the PCR Samples

• Use the worksheet table as a guide.
• Coordinate sample numbering across the class to avoid duplicate or confusing labels.
• In the example, the group is assigned samples 5–9.
• Record each sample name and the forward and reverse primers used. The same primer pair is used for all samples in the example.

3. Gather and Handle the Reagents

The common reagents are:

• Molecular biology water
• Forward primer
• Reverse primer
• RedTaq Ready Mix

Check that the primers are marked for PCR and are at the appropriate concentration. Thaw the RedTaq Ready Mix before use, then keep it on ice to limit enzyme degradation.

4. Prepare the Cocktail

1. Label a fresh tube “cocktail.”
2. Use a micropipette appropriate for each volume.
3. Add the largest-volume reagents first where practical. In this procedure, add the water first so the RedTaq can remain on ice as long as possible.
4. Add the primers, pipetting small volumes directly into the larger volume to reduce reagent loss.
5. Add the RedTaq Ready Mix last, then return its stock to ice immediately.
6. Mix the cocktail.

5. Briefly Centrifuge

• Combine the cocktail tubes prepared by all groups in the class.
• Press and hold the centrifuge’s short button for a brief spin.

Speakers and Sources

• Instructional narrator: An unnamed speaker provides the protocol directions.
• Visual/source material: A worksheet and tables are referenced; no individual author or additional speaker is identified.