The Roche protocol recommends using only 10pg of plasmid DNA for probe labelling. As such, all three probes were diluted 1:10,000. A 1:1000 (999μL H2O + 1μL of plasmid) was made first. Then a 1:10 dilution was made (90μL H2O + 10μL from 1:1000 dilution of plasmid).
Additionally, I ran half reactions to conserve kit components. Roche recommends 50μL reactions; I ran 25μL and scaled all components appropriately.
All reactions were set up on ice and run in 0.2mL strip-cap PCR tubes.
Reaction calculations are here (Google Sheet): 20151109 – RLOv ISH Probe PCRs
- 95C – 5mins
- 95C – 15s
- 55C – 15s
- 72C – 30s
- Go to Step 2, repeat 39 times.
- 72C – 10mins
After the PCR, 5μL of each reaction was run on a gel.
Two reactions were run for each plasmid: one with the DIG label (indicated by a ‘+’) and one without (indicated by a ‘-‘). If the labeling was successful, the PCR products from those reactions containing DIG will be larger (i.e. migrate slower) than those without. That is exactly what we see in each of the three potential ISH targets.
So, we now have three ISH probes ready for action! Will proceed with making fresh ISH buffers and ISH.
Probes were transferred to 0.5mL snap cap tubes and stored in my -20C box.