Tag Archives: RLOv_membrane_gene_2

In-situ Hybridization (ISH) – RLOv Membrane Genes 1 & 2, Tail Fiber Gene: Day 1

To test out the viability of these RLOv ISH probes (from 20151109) and not waste black abalone slides if this doesn’t work, I selected three unstained red abalone post-esophagus sections:

RLO – NO PHAGE

  • 08:1-12-2
  • 08:1-12-3
  • 08:1-12-4

RLOv

  • 08:1-7-7
  • 08:1-7-8
  • 08:1-7-9

RLO STIPPLED – NO PHAGE

  • 08:1-15-7
  • 08:1-15-8
  • 08:1-15-9

All slides were processed in a single, horizontal glass slide incubator (200mL), unless otherwise noted.

All steps were conducted at room temperature (RT), unless otherwise noted.

DEPARAFFINIZATION & REHYDRATION

  • All slides were deparaffinized with three changes of xylene (SafeClear II; Fisher) for 10mins each.
  • Slides were hydrated with a graded ethanol series (100%, 100%, 80%, 70%, 50%) for 3mins each.
  • Slides were rinsed with molecular grade H2O.

PREHYBRIDIZATION

  • Tissue sections were equilibrated in Tris Buffer (0.2M Tris-HCl, 2.0mM CaCl, pH = 7.2) for 5mins.
  • Tissues were permeabilized for 1.5hrs in preheated 50ug/mL Proteinase K (Qiagen) in Tris Buffer @ 56C.
  • Slides were rinsed with 1x PBS three times, 10mins each.
  • Slides were incubated 30mins in 30mL Prehybridization Buffer (50% deionized formamide, 4x SSC) @ 53C in a cylindrical glass slide incubator due to limited volume of deionized formamide available:

  • Prepared probes by boiling 3mins and immediately incubating in ice water bath for 30mins.
  • Slides were rinsed with 2x SSC and air dried for 5mins.
  • Probes were diluted 1:300 in 1000uL of Prehybridization Buffer. All three negative control probes (indicated by “-C” in subsequent labeling) were combined into a single dilution.

NOTE: RLOv Membrane Gene 2 probe was ruined because boiling water got into the tube during denaturation. This didn’t happen to any of the other tubes that were all boiled at the same time. Not sure what happened. However, this may have worked out OK because I did not pull enough slides to accomodate the negative control probes. So, now that I’m not able to test three probes, I can use the negative control probes!

HYRBIDIZATION

  • 300uL of probe solutions and cover slip were added to the following slides:

  • The three groups of slides were placed into separate slide cases and a 1mL of Prehybridization Buffer was added to each case (to maintain high humidity during incubation).
  • The cases were incubated on their sides O/N @ 53C.
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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.

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PCR – RLOv Clones

Colony PCRs were performed on each of the transformations from 20151015 (RLOv_ DNA_helicase, RLOv_head_to_tail, RLOv_membrane_gene_1, RLOv_membrane_gene_2, RLOv_tail_to_fiber) to confirm successful ligations in plasmid pCR2.1 using the M13F/R vector primers.

Colonies were picked form the transformation plates with pipette tips, re-streaked on a secondary, gridded, numbered LBAmp100+x-gal plate and then used to inoculate the respective PCR reactions.

Six white colonies (positive clones) and a single blue colony (negative clone) were selected from each transformation.

Master mix calcs are here (Google Sheet): 20151019 – Colony PCRs RLOv

Restreaked plates were incubated @ 37C O/N and then stored @ 4C (Parafilmed).

30μL of each reaction was run on a 1% agarose 1x Low TAE gel, stained w/EtBr.

Results:

 

All the PCRs look good. All white colonies selected contain a PCR product of appropriate size (i.e. larger than the blue colonies; negative [-C] control). Will select clones #1 from each to grow up for plasmid prep.

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Cloning – Purified Abalone RLOv PCR Products

Purified PCR products from 20151008 (in situ hybridization [ISH] primers) & 20151009 (qPCR primers) RLOv primers were used for cloning. The qPCR primers are intended to develop a qPCR standard curve and the ISH primers are intended to develop three ISH probes.

  • RLOv_DNA_helicase (for qPCR)
  • RLOv_head_to_tail_gene (for qPCR)
  • RLOv_membrane_gene_1 (for ISH)
  • RLOv_membrane_gene_2 (for ISH)
  • RLOv_tail_fiber_gene (for ISH)

The PCR products were separately ligated using The Original TA Cloning Kit (Invitrogen).

LIGATIONS

REAGENT SINGLE REACTION VOL (μL) x 5.5
Purified PCR 5 NA
10x Ligase Buffer 1 5.5
pCR2.1 Vector 2 5.5
H2O 1 5.5
T4 DNA Ligase 1 5.5
TOTAL 10 22

Ligation reactions were set up on ice.Combined 5μL of purified PCR product with 5μL of ligation master mix in a 1.5mL snap cap tube. Incubated 24hrs @ RT.

 

TRANSFORMATIONS

50μL of X-gal (20mg/mL) was added to a LB-Amp100 plates, spread and warmed @ 37C.

Five vials of OneShot TOP 10 chemically competent cells (Invitrogen) were thawed on ice. 3μL of the ligation reaction was added to the cells, gently mixed and incubated on ice for 5mins. 50μL of cells were transferred to the LB-Amp100+X-gal plates, spread and incubated O/N at 37C.

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Agarose Gel – Phage ISH Primers PCRs

Ran PCR products from yesterday on a 1% agarose 1x TBE gel, stained with ethidium bromide.

Results:

IMPORTANT NOTE: The negative control sample should actually be labelled UW08:22-11A.

 

PRIMER SET EXPECTED PCR SIZE (bp) RESULT SIZE (bp)
RLOv_membrane_gene_1 401 ~400bp
RLOv_membrane_gene_2 318 ~400bp
RLOv_tail_fiber_gene 451 ~500bp

PCR looks great. Excellent amplification in the RLO positive samples (06:6-54), with no amplification in the negative controls (UW08:22-11A) nor in the no template controls (NTC).

Excised the bands from each of the RLOv positive samples (see gel image below) and purified the DNA using UltrafreeDA Spin Columns (Millipore) according to the manufacturer’s protocol. DNA was stored @ 4C for cloning/labelling/sequencing at a later date.

Gel image showing excised regions.

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PCR – New Withering Syndrome Phage ISH Primers

Ran a PCR using the new ISH primers that I previously designed:

  • RLOv_tail_fiber_gene
  • RLOv_membrane_gene_1
  • RLOv_membrane_gene_2

Template DNA was black abalone DNA (from digestive gland [Dg]): 06:6-54 (from 4/9/2008)

Negative control DNA: UW08:22-11A (from 3/5/2007)

No template controls (NTCs) were also run.

All samples were run in duplicate, in 0.5mL PCR tubes.

 

Master mix calcs

REAGENT SINGLE REACTION (μL) x6.6 (μL)
Template 1 NA
2x Apex Red Master Mix 12.5 82.5
Primer Forward 0.5 3.3
Primer Reverse 0.5 3.3
H2O 11.5 75.9
TOTAL 25 Add 24μL to each tube

 

Cycling Params (PTC-200; MJ Research)

STEP TEMP (C) TIME (s)
Initial Denaturation
  • 95
  • 600
40 Cycles
  • 95
  • 55
  • 72
  • 15
  • 15
  • 30

 

Samples were held O/N at 4C. Will run on gel tomorrow.

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