MycoLight™ vPCR350

Before beginning the experiment, thaw MycoLight™ vPCR350 at room temperature and briefly centrifuge to collect the dried pellet.

1. Prepare bacterial samples.

2. Add MycoLight™ vPCR350 dye to bacterial samples.

3. Incubate samples for 20 minutes exposed to light (Long wavelength 365 nm).

4. Centrifuge samples to remove any excess dye.

5. Extract genomic DNA from bacterial samples.

6. Perform PCR/qPCR using appropriate primers and master mix.

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

1. To prepare a 5 mM MycoLight™ vPCR350 stock solution, add 300 μL of DMSO to the vial of MycoLight™ vPCR350.

   Note:    Any unused MycoLight™ vPCR350 stock solution should be divided into single-use aliquots and stored at ≤ -20 º C, protected from light. Avoid repeated freeze-thaw cycles.

For optimal results, it is recommended to conduct the experiment in low-light settings to reduce the impact of light on MycoLight™ vPCR350.

1. Inoculate the desired bacterial samples in a suitable media broth. Culture overnight until the OD600 reaches ~1.

   Note: This protocol is tailored for a 500 µL volume sample. Adjust the volume of the bacterial sample according to the experiment's scale.

2. Optional: To prepare dead cell controls, heat the bacterial sample (500 uL in a tube) at 90°C for 5 minutes.

3. Aliquot 500 μL of bacterial culture into individual clear microcentrifuge tubes. For each sample, prepare one tube for MycoLight™ vPCR350 treated cells and another for untreated cells (without MycoLight™ vPCR350 dye added) to calculate dCt values.

4. Add 5 µL of MycoLight™ vPCR350 stock solution to the bacterial samples.

   Note: Adding 5 µL of stock solution will result in a final dye concentration of 50 µM. This concentration can be used as a starting point and further optimization may be necessary to achieve optimal results.

5. Expose the samples to 365 nm light using the long settings in the instrument to cross-link the samples with MycoLight™ vPCR350 to DNA.

   Note: For optimal activation, use a 3-UV lamp with wavelengths of 254/302/365.

   Note: If samples are generating heat, incubate them on ice.

6. Centrifuge samples at 5000 x g for 10 minutes. Then remove the supernatant without disturbing the cell pellet.

7. Extract genomic DNA using your preferred method or a commercially available kit suitable for the sample type.

8. Perform qPCR using primers targeting a specific genomic DNA sequence of your choice.

   Note: Perform qPCR using the same volume for all samples. Normalization of the concentration of DNA is not required.

1. After completing the qPCR, calculate the Ct (Threshold Cycle) value for each sample.

2. To determine if MycoLight™ vPCR350 has sufficiently inhibited the amplification of DNA in dead cells, calculate the delta Ct (dCt) for each of your control cells using the formulas below:

   • dCt (Live) = Ct (Live MycoLight™ vPCR350 treated sample) – Ct (Live untreated)
   • dCt (Dead) = Ct (Dead MycoLight™ vPCR350 treated sample) – Ct (Dead untreated)

3. For the live cell control, the expected result should be close to 0. A greater difference between dead and live cells suggests that the MycoLight™ vPCR350 treatment has effectively inhibited DNA in the dead cell samples.