Sean Lauber:qRT-PCR (TaqMan): Difference between revisions
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4. Add 5 μL of 2 ng/μL cDNA 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 | 5. Seal plate with sticky film, spin down, and place into the 7900 HT qPCR machine[[Lauber:Using the 7900 HT qPCR Machine]] | ||
Revision as of 08:37, 30 March 2012
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 as well as controls NTC and NAC) 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 (orange), B will probe for IL-6 (green) and C will probe for OSM (purple). 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 7900 HT qPCR machineLauber:Using the 7900 HT qPCR Machine
