Tag Archives: Ultrafree-DA

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.


PCR – RLOv for Cloning & Sequencing

After yesterday’s confirmation that the qPCR primer/probe sets for RLOv DNA helicase and head-to-tail were functional and specific for the RLOv, I needed to generate PCR products to clone and sequence.

Primers tested:

  • RLOv_DNA_helicase
  • RLOv_head_to_tail_gene

Template DNA:

  • 06:6-54

All samples were run in duplicate.

Master mix calcs are here: 20151009 – PCR RLOv

Cycling Params (PTC-200; MJ Research)

Initial Denaturation
  • 95
  • 600
40 Cycles
  • 95
  • 55
  • 72
  • 15
  • 15
  • 30

Samples were run on a 0.8% agarose 1x TBE gel, stained with ethidium bromide.


Amplification looks great. No amplification in no template controls (NTCs). Excised bands and purified products using Ultrafree DA Spin Columns (Millipore). Samples will be stored @ 4C until I am able to clone them for sequencing.


Gel image showing excised bands. And, it’s a complete hack job, which is embarrassing…


Agarose Gel – Phage ISH Primers PCRs

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


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


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.


Gel Purification – Olympia Oyster and Sea Pen PCRs

Purified DNA from the remaining PCR bands excised by Jake on 20150609 and 20150610, as well as Jonathan’s sea pen PCRs from 20150604, using Ultrafree-DA spin columns (Millipore). Transferred gel pieces from storage tubes (1.5mL snap cap tubes) to spin columns. Spun 10,000g, 5mins @ RT. Transferred purified DNA back to original storage tubes. See the sequence_log (Google Sheet) for a full list of the samples and the sequencing plates layouts. Purified DNA was stored @ 4C O/N. Will prepare and submit plates for Sanger sequencing tomorrow.


PCR – Ireland Clam RLO DNA S/6/14 #19 (from 20150130)

After previously confirming that the issue with previous PCRs was due to bad reagents, I re-ran the PCR on the clam RLO DNA isolated 20150130 using a set of universal 16s primers, as well as a universal 18s primer set to serve as a positive control that amplifiable DNA was present in the sample.

Master mix calcs are here: 20150219 – cPCR Universal Primers Apex Red MM

Primers being used are:

  • 16s/23s-F/R
  • 27F, 1492R
  • EHR16D, EHR16R (universal ehrlichia)
  • EUB-A/B
  • 18s EUK 581 F, 18s EUK 1134 R

Cycling params were:
1 cycle of:

  • 95C – 10mins

40 cycles of:

  • 95C – 15s
  • 50C – 15s
  • 72C – 1mins

Samples were run on 1.0% agarose, low TAE gel stained w/EtBr.


Ladder used was O’GenRuler 100bp DNA Ladder (Thermo-Fisher).

No sample was loaded directly next to ladder to facilitate excision, if necessary.

Each sample was accompanied by a no template control (NTC).

The ehrlichia universal primers (EHR) and the universal 18s (18s) primers are the only two primer sets that do not have contamination present in the NTCs.

Excised the EHR band and purified with Ultrafree-DA columns (Millipore). Purified DNA was stored @ -20C and will be used for cloning/sequencing next week.

Have already ordered additional primer sets of those above that are contaminated. Will re-run the PCR with those new, sterile primer sets when they arrive to obtain a larger product (the EHR amplicon is only ~350bp).


PCR – Withering Syndrome Phage

We received MiSeq data back from Stan Langevin (samples submitted 20140717) and he believes he has sequenced the entire WS phage. Carolyn and Colleen designed some primers on two of the open reading frames annotated by Stan. Ran PCR with the three primer sets to test out:

  • 1_ORF25F_225_CSF, 1_ORF25R_399_CSF
  • 2_ORF25_121_CAB, 2_ORF25R_320_CAB
  • 3_ORF20F_121_CSF, 3_ORF20R_326_CSF

Master mix calcs are here: 201400813 – PCR WS phage

Cycling params:

Ran samples on 1.2% 1x TBE + EtBr.


Ladder: O’GeneRuler 100bp DNA Ladder (ThermoFisher)

Good amplification from all three primer sets. The pinto abalone sample (UW08:22-65) that should be naive for withering syndrome and phage did not amplify as expected.

Excised bands from each primer set in the 06:6-41 group and purified using Ultrafree DA spin columns (Millipore). Will save for potential cloning usage, depending on future results.


PCR – Hexokinase and Partial Exon #1

Performed PCR using newly designed primers to amplify the C. gigas hexokinase “promoter” (-2059bp from start) along with a portion of the first exon.

Primers used were Cg_Hk_Prom_pBAD_-2059 (SRID: 1518) and Cg_HK_Exon1_R (SRID: 1520).

Template used was C.gigas gDNA BB15 (from 20090519; 0.4216ug/uL). Master mix calcs are here. Cycling params are the same used on 20130227.

Samples were run in duplicate.


Lane 1: Hyperladder II (Bioline)

Lanes 2-3: C.gigas gDNA

Lanes 4-5: NTCs

We see a band of >2000bp (that’s the maximum on the molecular weight marker). The bands from each replicate were excised, purified using Ultrafree-DA columns (Millipore) and stored at 4C.


PCR – Region Outside of COX/PGS qPCR Primers

Ran PCR using primers Cg_COX_982_F and Cg_COX_2138_R (SR IDs: 1149 & 1151, respectively). Template was pooled cDNA from 20110311 of various C.gigas tissues. These primers anneal 5′ and 3′ of where the qPCR primers for both COX1/PGS1 and COX2/PGS2 anneal. Master mix calcs and cycling params are here. Ran multiples of the same reaction to ensure sufficient product for use in cloning/PCR.


Gel is loaded with Hyperladder I (Bioline) and 7 samples (no NTC; don’t ask). Band in each lane is of the expected size (~1200bp). Each band was excised and purified using Ultra-free DA columns (Millipore), according to protocol. Purified DNA will be used in a subsequent PCR using the qPCR primers for COX/PGS 1&2 BEFORE cloning this product for sequencing.


PCR – Full-length PGS1 cDNA

Still have insufficient quantities of DNA for sequencing. Master mix calcs and cycling params are here. Additionally, used some of the purified PCR product as template in one of the reactions, just for comparison purposes. cDNA template was pooled cDNA from 20110311 from various C.gigas tissues. Also, increased the amount of template 4-fold in an attempt to obtain higher yields of PCR products for sequencing.


Lane 1: Hyperladder I (Bioline)

Lane 2: PCR 1 (cDNA template)

Lane 3: PCR 2 (cDNA template)

Lane 4: PCR 3 (PCR template)

Lane 5: Neg. Control

Bands were excised and will be purified using Ultra-free DA columns (Millipore). Also, it’s very clear that using the purified PCR product as template produced a much greater yield, although there appear to be some spurious, high-molecular weight banding/smearing.


PCR – Full-length PGS1 & PGS2 cDNAs

Ran PCR to amplify full-length cDNAs of PGS1 & PGS2 (COX1 & COX2) using primers designed to anneal in the 5’/3’UTRs of each isoform. PGS1 primers = SRIDs: 1377, 1378. PGS2 primers = 1376, 1375. Master mix calcs and cycling params are here. cDNA was pooled cDNA made 20110311 from various tissues.

PGS1 Expected Size = ~2300bp

PGS2 Expected Size = ~2500bp



Lane 1 – Hyperladder I (Bioline)

Lane 2 – PGS1

Lane 3 – PGS1 NTC

Lane 4 – PGS1 NTC

Lane 5 – PGS2

Lane 6 – PGS2 NTC

Lane 7 – PGS2 NTC

PGS1 Results: PGS1 PCR produces a single band of the expected size (~2300bp), indicating that the two primers, which were designed to anneal in the 5’/3’UTRs of the gene and should be highly specific to just this isoform, work perfectly. The band was excised and stored @ -20C in “Sam’s Miscellaneous” box.

PGS2 Results: PGS2 PCR didn’t produce any product. Will repeat with a lower annealing temp (50C instead of 55C).