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

Ligation reactions:

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

Incubate 1hr @ RT.

TRANSFORMATION

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.

Results:

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.

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).

Results:
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.

Sanger Sequencing – pCR2.1/OsHV-1 ORF117 Sequencing Data

Received the Sanger sequencing data back from Genewiz for the samples I submitted last week.

AB1 files were downloaded as a zip file and stored in the Friedman Lab server: backupordie/lab/sequencing_data/Sanger/30-19717124_ab1.zip

Files were analyzed using Geneious 10.2.3.

Geneious analysis was exported (compatible with version 6.0 and up) and saved to the Friedman Lab server:

backupordie/lab/sam/Sequencing_Analysis/Sanger/20170821_oshv_orf117_sanger.geneious

Results:

After vector ID and trimming, all sequences from both colonies were aligned, resulting in an 867bp contig. The size of this contig jives perfectly with the bright PCR band at ~1000bp I saw when screening the two colonies (the ~1000bp includes 300bp of vector sequence from using the M13 primers).

 

The alignment above shows that there were no gaps in the sequencing between the two sequencing primers (M13 forward and M13 reverse). I point this out because the insert in this plasmid was supposed to be the full-length OsHV-1 ORF117 (which is ~1300bp), as described in: Detection of undescribed ostreid herpesvirus 1 (OsHV-1) specimens from Pacific oyster, Crassostrea gigas. Martenot et al. 2015. As the sequencing shows, that is not what is cloned in this vector.

To determine what was actually cloned in this vector, I performed a BLASTx against the nr database, using the consensus sequence generated from the alignment above:

 

BLASTx generated a total of six matches, five of which match OsHV-1 ORF117 (the hypothetical and RING finger proteins listed above actually have alternate accession numbers that all point to ORF117). However, notice in the one alignment example provided at the bottom of the above image, the Query (i.e. our consensus sequence) only starts aligning at nucleotide 109 and matches up with the NCBI OsHV-1 ORF117 beginning at amino acid 158.

The results clearly show that the insert in this vector is OsHV-1 ORF117, but it is not the entire thing. To confirm this, I aligned the consensus sequence to the OsHV-1 genome (GenBank: AY509253.2) using Geneious:

 

In the image above, I have zoomed into the region in which our sequencing consensus aligned within the OsHV-1 genome. In order to see in more detail, please click on the image above. There are two noticeable things in this alignment:

  1. The insert we sequenced doesn’t span the entire ORF117 coding sequence (the yellow annotation in the image above).

  2. There’s a significant amount of sequence mismatch (112bp; indicated by black hash marks) between the sequenced insert and the OsHV-1 ORF117 genomic sequence from GenBank, at the 5′ end of the insert.

Will pass this info along to Carolyn and Tim to see how they want to proceed.

Plasmid Isolation – pCR2.1/OsHv-1_ORF117 Miniprep

The last run at this failed, but I think that was due to old ampicillin stocks; leading to no selective pressure for transformants that actually contained plasmid.

I’ve since remedied that.

Grew up 5mL of culture from the only two transformants in 1xLB + 100ug/mL of (fresh!) ampicillin @ 37C on a rocking platform in a 15mL conical over night (~18hrs).

Isolated plasmid DNA from the entire 3mL of culture (repeated pelleting of bacteria in the same 1.5mL snap cap tube) using the QIAprep Spin Miniprep Kit, according to their protocol.

Eluted DNA with 50uL of EB Buffer.

Quantified on the Roberts Lab Qubit 3.0 using the dsDNA BR Kit (broad range) and 10uL of sample.

Results:

Quantification (Google Sheet): 20170817_quantification_oshv_orf117_plasmid

Colony 1 – 130ng/uL
Colony 2 – 148ng/uL

Yields look perfect. Will submit for sequencing at Genewiz (they need 10uL of ~50ng/uL DNA) and see what we have here…

PCR – pCR2.1/OsHV-1_ORF117 Colony Screens

Performed PCR with M13 vector primers on the two colonies that grew from yesterday’s transformation.

Master mix calcs:

2x Apex Red Master PCR Mix: 33uL
M13 forward: 1.5uL
M13 reverse: 1.5uL
H2O: 29.7uL

Added 20uL to each PCR tube (0.2mL PCR strip tubes).

Bacteria was collected from each colony with a sterile 10uL pipet tip, which was used to streak on a separate LB Amp100 plate and then introduce bacteria to the appropriate PCR tube.

Cycling params (PTC-200 MJ Research):

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.

Results:

 

 

Well, this might seem promising, due to the intensity of that band (~1000bp). A band of that size was also produced the last time, ableit with much less intensity.

The very bright, 1000bp band generated from Colonies 1 (left) and 2 (right) is not the expected size. Based on this paper (Detection of undescribed ostreid herpesvirus 1 (OsHV-1) specimens from Pacific oyster, Crassostrea gigas. Martenot et al. 2015), the insert size should be ~1300bp (Tim Green indicated he used the primers listed in the paper to clone ORF117).

However, there is a less bright band just above 1500bp. Oddly, this would be the expected size for this PCR (1300bp insert + 200bp of vector sequence from the M13 primers). The lower intensity is discouraging, though, because this indicates that M13 primers are preferentially binding whatever is producing that 1000bp band.

Regardless, I’ve already inoculated two liquid cultures to grow up over night. I’ll perform a plasmid isolation on them tomorrow morning. Hopefully they actually yield some plasmid DNA to do some work with, unlike last time.

Transformation – pCR2.1/OsHV-1_ORF117 into One Shot Top10 Chemically Competent Cells

Yesterday’s transformation with freshly prepared ampicillin didn’t produce any transformants, suggesting the DNA concentration is too low.

Previously, I tried to elute the DNA from one of the spots Tim sent with 50uL. This volume was enough to soak the Whatman paper and produce excess liquid. In retrospect, I think the volume was too large and diluted the DNA too much (concentration wasn’t measurable via Qubit)

Today, I eluted with 25uL. Since this volume was too little to produce excess liquid, I created a spin “filter” to extract the absorbed liquid. Briefly, I punctured the top and bottom of a 0.5mL snap cap tube with an 18 gauge needle, inserted the Whatman paper disc into this tube, and then put this tube in a 2mL snap cap tube. This assembly was spun @ 18,000g RT for 3 mins.

Used 5uL of the pCR2.1/OsHV-1_ORF117 plasmid provided by Tim Green to transform a single aliquot of One Shot Top10 Chemically Competent Cells (Invitrogen), according to the “Rapid Transformation” protocol (thaw cells on ice, add DNA, incubate 5mins, plate on pre-warmed ampicillin plates).

Cells were plated on pre-warmed (37C) LB Amp100 plates.

Plates were incubated overnight at 37C.

Results:

Wow, only two colonies! Well, as they say, you only need one. Will PCR, re-streak, and inoculate 5mL liquid cultures to see if either of these colonies seem to have the insert.

Transformation – pCR2.1/OsHV-1_ORF117 into One Shot Top10 Chemically Competent Cells

This is a repeat since the previous attempt at obtaining sufficient quantities of plasmid for sequencing failed. Although I’m not sure why, I figure it’s easy enough to re-do using ampicillin stocks that aren’t many years old. :)

The old ampicillin may not have been strong enough to put enough selective pressure on transformants, which possibly led to such little plasmid recovery.

I prepared fresh ampicillin solution (20mg/mL) and made new LB plates (ampicillin concentration 100ug/mL).

Used 5uL of the pCR2.1/OsHV-1_ORF117 plasmid provided by Tim Green to transform a single aliquot of One Shot Top10 Chemically Competent Cells (Invitrogen), according to the “Rapid Transformation” protocol (thaw cells on ice, add DNA, incubate 5mins, plate on pre-warmed ampicillin plates).

Cells were plated on pre-warmed (37C) LB Amp100 plates.

Plates were incubated overnight at 37C.

Results:

No transformants. So, this suggests that the original ampicillin was bad. Now, the lack of transformants suggests the plasmid concentration is too low. Will try eluting the DNA from the second spot of Whatman paper.

Plasmid Isolation – pCR2.1/OsHv-1_ORF117 Miniprep

Grew up 5mL of culture from re-streaked colony #1 in 1xLB + 100ug/mL of ampicillin @ 37C on a rocking platform in a 15mL conical over night (~18hrs).

Isolated plasmid DNA from the entire 5mL of culture (repeated pelleting of bacteria in the same 1.5mL snap cap tube) using the QIAprep Spin Miniprep Kit, according to their protocol.

Eluted DNA with 50uL of EB Buffer.

Quantified on the Roberts Lab Qubit 3.0 using the dsDNA BR Kit (broad range) and 1uL of sample.

Results:

The results are not good. Using 1uL of the sample, I received an error message that the concentration was out of range – too low!

Repeated, but used 10uL of sample. Concentration was displayed as 1.13ng/uL!!

This is insufficient yield/concentration for sequencing.

It’s possible that the kit is too old (no receipt date marked on the box…)? The reagents shouldn’t go bad, but can the columns? I feel like the resins in the columns are pretty stable, just like the various buffers.

The ridiculously low yields could also possibly indicate that the bacteria don’t actually have the plasmid, but PCRs from yesterday suggest otherwise.

Maybe the column was overloaded? I’ll repeat this next week, but using smaller culture size and/or not using the column and perform an isopropanol precipitation instead…

And/or make fresh stock of ampicillin (current stock is many years old, but has been frozen).