I replaced the alarm battery for each of our -80C freezers – Revco and Sanyo.
Revco battery – Fisher NC1397422
Sanyo battery – Fisher NC1406075
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
Both standard curves are acceptable (see images below).
No amplification with either primer/probe set in the following samples:
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
Purified OsHV-1 ORF117 PCRs from earlier today were separately ligated using the Original TA Cloning Kit (Invitrogen).
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.
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:
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:
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
Carolyn had expressed interest in sequencing these.
I ran conventional PCRs using the ORF117 primers found in:
Template DNAs were:
Aus A (Australian)
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
Cycling params were:
95C – 10mins
95C – 15s
55C – 15s
72C – 90s
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.
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).
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
See labeled amplification plots below.
Received genomic DNA from oysters infected with OsHV-1 variants.
Received the following samples with Australian (Aus) and French (M) variants from Carolyn (stored in my -20C Box #2):
Received the following samples with California variant from Natalie Rivlin (University of Maryland) (stored in -20C in FSH 240):
Designed primers to target highly variable region (5′ end) of the partial Australian OsHV-1 ORF117 sequence that we have.
Primers were designed using Primer3.
Have ordered from IDT and will test once I get other OsHV-1 variants – to check specificity.
Slides were stashed in FSH 236 in Cabinet 24 with the rest of the lab’s slides.
Quantified DNA extractions from Ava’s samples that I isolated earlier this month, as well as some older samples that I hadn’t quantified yet.
Used the Roberts Lab Qubit 3.0 and the Qubit dsDNA BR Kit (broad range). Used 5uL of template for the first and third groups and 1uL of template for the second group (see Results below).
All data was added to the master extraction spreadsheet (Google Sheet): ava_abalone_master_extraction_list