Albina wants to screen her isolated colonies to determine whether they contain resistance gene A. She picks 5 colonies, lyses them at 95ºC, and sets up a PCR reaction using appropriate primers. These primers should produce a 4 kb amplicon if resistance gene A is present. She has primer stocks at 100 µM and a diluted version at 5 µM. She also has a bacterium with a plasmid known to contain resistance gene A.

How should she set up her PCR? Describe how to make the master mix.

- Template DNA: 1.0 µL
- Primer 1 (0.25 µM): [specify volume]
- Primer 2 (0.25 µM): [specify volume]
- 2X PCR master mix: [specify volume]
- Water: [specify volume]

Total: 50.0 µL

Albina should create a PCR master mix for each reaction that includes 0.5 uL of each primer at 5uM concentration, 1.0 uL of template DNA, 25 uL of 2X PCR master mix, and 23 uL of water, totalling 50 uL.

Explanation:

To set up a PCR to determine if the isolated colonies have the resistance geneA, Albina should begin by preparing a master mix that includes the primers, PCR master mix, and water. Since the final concentration of each primer in the PCR should be 0.25uM, and the total volume is 50uL, Albina needs to dilute her 100uM stock primer solutions. She can use the 5uM diluted versions of the primers for this purpose.

Creating a Master Mix for One Reaction

Add 1.0 uL of DNA template from the bacterial colonies.
Pipette 0.5 uL of Primer 1 (from her 5uM stock) and 0.5 uL of Primer 2 (from her 5uM stock), reaching 0.25uM final concentration for each primer in the 50uL reaction volume.
Add 25 uL of 2X PCR master mix—this will provide the nucleotides, Taq polymerase, and other necessary components.
Top up the reaction with 23 uL of water to bring the total reaction volume to 50uL.
The 2X PCR master mix contains twice the required concentration of nucleotides and Taq polymerase so that when it is added to an equal volume of other components, the correct concentrations are achieved in the final PCR reaction mixture.

PCR Cycling Conditions:

Initial denaturation at 95°C for 2 minutes.
35 cycles of:
95°C for 30 seconds (Denaturation).
55°C for 30 seconds (Annealing).
72°C for 1 minute (Extension).
Final extension at 72°C for 10 minutes.