Step 3 – synthesis of 3,4-difluorophenylethan-1-ol

Step 3.jpg

Masses and measurements:

3,4-difluorophenyl acetic acid - 0.9934g

LiAlH4 - 0.2386g

THF - 6.3mL


The 3,4-difluorophenyl acetic acid (0.9934g, 5.78mmol) was added to a RBF and was dissolved in THF (6.3mL). The reaction mixture was further stirred whilst the LiAlH4 (0.2386g, 6.29mmol) was added and the reaction mixture was stirred under reflux for 2-3 hours or until TLC analysis (1:1 ethyl acetate & petroleum ether) confirmed the completion of the reaction. Around 20mins after all reagents were added, the mixture turned a deep red and soon after turned to a pale yellow solution.

reaction of alcohol.jpg
alcohol synthesis.jpg

Once the reaction was complete, the reaction mixture was left to cooland quenched with 25% NaOH (10mL) and the product was seperated and extracted using 2M HCl (20mL) and ethyl acetate (20mL). Seperation of the reaction mixture gave 2 colourless solution layers. The aqueous layers were re-extracted using diethyl ether (20mL) - it was learnt in the research we underook that the alcohol product is very volatile so diethyl ether was used as it has a lower volatilty to match that of the final product. The organic layers were collected, dried using MgSO4 and filtered under vacuo. The filtrate was rotary evporated with a small amount of solvent present still. The product given was a pale yellow oily solution (0.729g, 79.85%).

qPCR – Pinto Abalone DNA with WSN1 and RLOv DNA Helicase

Ran qPCRs using both WSN1 and RLOv DNA Helicase primers on the pinto abalone DNA isolated earlier today, as well as one additional sample that Sean Bennett had previously isolated DNA from: 15:6-26E

RLOv helicase standard curve is from 20151106.

WSN1 standard curve is p18RK7 from 20170703

All samples were run in duplicate.

Master mix calcs (Google Sheet): 20171226 – qPCR Pinto WSN1 & RLOv DNA Helicase.

Plate layout, cycling params, etc. can be seen in the qPCR Reports (see Results below).


RLOv helicase qPCR Report (PDF): Sam_2017-12-26 13-19-51_CC009827_RLOv_helicase.pdf
RLOv helicase qPCR File (CFX): Sam_2017-12-26 13-19-51_CC009827_RLOv_helicase.pcrd

WSN1 qPCR Report (PDF): Sam_2017-12-26 13-19-51_CC009827_WSN1.pdf
WSN1 qPCR File (CFX): Sam_2017-12-26 13-19-51_CC009827_WSN1.pcrd

Both standard curves are acceptable (see images below).

No amplification with either primer/probe set in the following samples:

  • 15:30-01
  • 15:30-04

I believe these are both “Control” samples (i.e. unexposed) and no amplification was expected.

All other samples amplify. See qPCR Reports for copy numbers.

RLOv Helicase Amplification & Standard Curves

WSN1 Amplification & Standard Curves

Cloning – Purified OsHV-1 ORF117 PCRs

Purified OsHV-1 ORF117 PCRs from earlier today were separately ligated using the Original TA Cloning Kit (Invitrogen).


Ligation reactions:

  • PCR product: 5μL
  • 5x Buffer: 2μL
  • Vector (pCR2.1): 2μL
  • T4 Ligase: 1μL

Incubate 1hr @ RT.


50μL of X-gal (40mg/mL) was added to a LB-Amp100 plate, spread and warmed @ 37C.

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


All three transformations failed. All of them produced only blue colonies and very few total colonies.

The low number of colonies prompted me to look at the troubleshooting in the manual for The Original TA Cloning Kit (Invitrogen). It turns out that after six months of storage, the vector begins to lose the T overhangs. The kit I used is from 2014; three years beyond the tentative expiration date. This is likely the cause of the failed transformations.

DNA Isolation & Quantification – Pinto Abalone

Isolated DNA from the following pinto abalone (Haliotis kamtschatkana) digestive gland tissues (stored in ethanol), collected by Sean Bennett as part of his Capstone project:

Accession Weight(mg)
15:30-01   194
15:30-04   67
15:31-01   34
15:31-02   107
15:31-03   83
15:31-04   80

Tissue was weighed and then DNA extracted.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

  • Samples were briefly homogenized (due to their stiffness resulting from ethanol fixation) in the InhibitEX Buffer using disposable plastic pestles.
  • Homogenized tissue was incubated at 95C to maximize cell lysis
  • Followed “human DNA analysis” protocol for remainder of protocol (to maximize sample recovery)
  • Eluted DNA with 100μL Buffer ATE

Used the Roberts Lab Qubit 3.0 and the Qubit hsDNA Kit (high sensitivity). Used 1uL of template for all samples.

Samples were stored at -20C in FSH240 in the “Pinto Transcriptome DNA” box.


All samples have DNA.

Concentrations (Google Sheet): 20171226_qubit_DNA_pinto_ab

PCR – OsHV-1 ORF117 from Australian, California, & French Variants

Carolyn had expressed interest in sequencing these.

I ran conventional PCRs using the ORF117 primers found in:

Genome exploration of six variants of the Ostreid Herpesvirus 1 and characterization of large deletion in OsHV-1μVar specimens. Martenot et al. 2013


Template DNAs were:

Aus A (Australian)
M1 (French)
TB15-15-305 (Californian)

All three template DNA samples were received from Carolyn/Colleen on 20171221. Used 2uL of 1:100 dilutions from each stock.

Master mix (25uL reactions)

2x Apex Red Master PCR Mix: 27.5uL
M13 forward: 1.1uL
M13 reverse: 1.1uL
H2O: 20.9uL

Cycling params were:

1 cycle:

95C – 10mins

30 cycles:

95C – 15s
55C – 15s
72C – 90s

1 cycle:

72C – 10mins

PCR reactions were run on a 1% agarose 1xTBE gel + EtBr.

5uL of O’GeneRuler DNA Ladder Mix was loaded for sizing.


The results are pretty interesting (but maybe not too helpful)!

Firstly, all three variants produced three different size products:

Aus A (Australian) – ~900bp
M1 (French) – ~1300bp
TB15-15-305 (Californian) – ~800bp

Of note, is that the paper from which these primers originated from, indicated that the PCR product generated was ~1300bp. The strain that that paper used for sequence analysis was the French strain (i.e. microVar)!

The other two strains amplified perfectly well, but are significantly smaller in size. This suggests a major deletion of some sort in ORF117 between the Australian/Californian vs. the French strain!

It also helps explain the discrepancy noted when we originally received the Australian ORF117 from Tim Green. He indicated his lab used the primers from the paper linked above and that the insert size was 1300bp. However, when I sequenced the ORF117 plasmid he sent to us, there was only 837bp of sequence (which would match the size of the product generated here, using the ORF117 primers from the paper)!

All bands were excised and DNA was purified using Ultrafree-DA spin columns (Millipore). I’ll clone all three and send of for sequencing.

qPCR – Test Australian OsHV-1 ORF117 Primers

Using primers I previously designed, I tested them out for functionality (using the clone #1 plasmid prep DNA I made previously) and specificity (using the Australian, California, & French variants recently received)

Created a working 1:100 dilution of ALL DNA tested here.

All samples were run in duplicate.

Master mix calcs are here (Google Sheet): 20171221 – qPCR Austrailian OsHV-1 ORF117 Primer Test

Cycling params, plate layout, etc. can be viewed in the qPCR Report (see Results below).

qPCR Report (PDF): Sam_2017-12-21 15-09-49_CC009827.pdf
qPCR Data File (CFX): Sam_2017-12-21 15-09-49_CC009827.pcrd

Firstly, the primers work and generate a single melt curve peak (see melt curve plot below); so that proves functionality.

Results are interesting.

Australian samples (plasmid and DNA) amplify.

French samples (M1 & M2) do not amplify.

California samples: 3 of 4 samples amplify.

It’s possible that the California sample that did not amplify is due to too little DNA present in the 1:100 dilution I used (or, possibly no DNA is present at all). I have not quantified the DNA in these samples – went off assumption that the samples had previously been confirmed to have DNA in them by the source laboratories.

Regardless, the primers used here will amplify the French variant, but will amplify Australian and Californian variants.

See labeled amplification plots below.

Synthesis of 5-(3,4-difluorophenethoxy)-3-(4-(pipperidin-1-yl)phenyl)-[1,2,4]triazolo[4,3-a]pyrazine


VTM010 (0.05 g, 0.12 mmol) was dissolved in degassed toluene (2 mL). Piperidine (0.025 g, 0.29 mmol), NaOtBu (0.016 g, 0.168 mmol), JohnPhos (1.6 mg, 5.4 μmol) and Pd 2 (dba) 2 (0.3 mg, 0.6 μmol) were added to the solution and stirred at 100°C for two weeks. The reaction was quenched with 2M HCl and the organic layer extracted (EtOAc 3x2 mL). The crude was concentrated under reduced pressure and purified via column chromatography (100% EtOAc).

VTM012 COLUMN.jpeg

Synthesis of 5-(3,4-difluorophenethoxy)-3-(4-(pyrrolidin-1-yl)phenyl)-[1,2,4]triazolo[4,3-a]pyrazine


VTM010 (0.05 g, 0.12 mmol) was dissolved in degassed toluene (2 mL). Pyrrolidine (0.02 g, 0.288 mmol), NaO t Bu (0.016 g, 0.168 mmol), JohnPhos (1.6 mg, 5.4 μmol) and Pd 2 (dba) 2 (0.3 mg, 0.6 μmol) were mixed in this order and stirred for two weeks at 100°C. The reaction was quenched (2M HCl). The organic layer was extracted (3x2mL EtOAc). The crude was concentrated in vacuo and purified via column chromatography (EtOAc).


NMR analysis suggested that a final product was not formed.