PCR – In-situ Hybridization (ISH) Probe

Ran probe-labeling PCRs to use in in-situ hybridization (ISH). Generated PCR probes for the following:

Phage ORF20

Phage ORF25

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.

Cycling Params:

  1. 95C – 5mins

  2. 95C – 15s

  3. 55C – 15s

  4. 72C – 30s

  5. Go to Step 2, repeat 39 times.

  6. 72C – 10mins

Ran 5uL of each sample on a 1% agarose 1x TBE gel, stained with EtBr.

Results:

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.