PCR – RLOv In-situ Hybridization (ISH) Probes

Ran probe-labeling PCRs to use in in-situ hybridization (ISH) using the PCR DIG Probe Sysnthesis Kit (Roche). Generated PCR probes for using the following BamHI-linearized plasmids:

  • pCR2.1/RLOv_membrane_gene_1
  • pCR2.1/RLOv_membrane_gene_1
  • pCR2.1/RLOv_tail_fiber

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

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

After the PCR, 5μL of each reaction was run on a gel.

Results:

Hyperladder I (Bioline)

PCR DIG probe labelling products run on 1.1% agarose 1x TBE gel stained w/EtBr. A ‘+’ indicates DIG reaction, while a ‘-‘ indicates no DIG in reaction.

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.

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.