Ran probe-labeling PCRs to use in in-situ hybridization (ISH). Generated PCR probes for the following:
Withering Syndrome (p18RK7, 3e6)
PCR calculations are here: 20141008 – ISH Probe PCRs
Used the PCR DIG Probe Synthesis Kit (Roche), with 10pg of template for each probe. Ran a DIG positive and DIG negative sample for each sample.
- 95C – 5mins
95C – 15s
55C – 15s
72C – 30s
Go to Step 2, repeat 39 times.
72C – 10mins
Ran 5uL of each sample on a 1% agarose 1x TBE gel, stained with EtBr.
Ladder: Hyperladder I (Bioline)
ORF25 and p18RK7 PCRs seemed to have worked as expected. This is evidenced by larger molecular weight bands in the DIG-positive samples (the DIG molecules incorporated during PCR result in slower migration through the gel).
However, the ORF20 PCR doesn’t seemed to have worked in either the DIG-postiive nor the DIG-negative. I won’t bother re-running this, since the ORF25 will also function as a probe for detection of withering syndrome phage.
Probes were stored @ -20C in my -20C box.