Sean Lauber:qRT-PCR (TaqMan)

I typically use 10 ng cDNA/Taqman reaction and I deliver 5 μL, so you want a cDNA concentration of 2 ng/μL. For this example I am going to pretend I have 2 samples (X and Y) and I want to probe for 18S, IL-6, and OSM. The 96-well template will look like this:

Row A will probe for 18S, B will probe for IL-6 and C will probe for OSM. So these are the probes you will include in those wells.

1. Dilute your cDNA to 2 ng/μL.

2. Prepare probe master mixes containing:

 12.5 μL universal master mix
  6.25 μL nuclease-free water
  1.25 μL PDAR (probe)

3. Add 20 μL of probe master mix to appropriate wells.

4. Add 5 μL of 2 ng/μL cDNA to appropriate wells.

5. Seal plate with sticky film, spin down, and place into the 5700 machine