Sam's Notebook

Projects

Miscellaneous

FastQC and Trimming - Metagenomics (Geoduck) HiSeqX Reads from 20180809

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Steven tasked me with assembling our geoduck metagenomics HiSeqX data. The first part of the process is examining the quality of the sequencing reads, performing quality trimming, and then checking the quality of the trimmed reads. It’s also possible (likely) that I’ll need to run another round of trimming. The process is documented in the Jupyter Notebook linked below. After these reads are cleaned up, I’ll transfer them over to our HPC nodes (Mox) and try assembling them.

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Installation - Microsoft Machine Learning Server (Microsoft R Open) on Emu/Roadrunner R Studio Server

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Steven recently saw an announcement that Microsoft R Open now handles multi-threaded processing (default R does not), so we were interested in trying it out. I installed MLR/MRO on Emu/Roadrunner (Apple Xserve; Ubuntu 16.04). Followed the Microsoft installation directions for Ubuntu. In retrospect, I think I could’ve just installed MRO, but this gets the job done as well and won’t hurt anything.

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Mox - Password-less SSH!

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The high performance computing (HPC) cluster (called Mox) at Univ. of Washington (UW) frustratingly requires a password when SSH-ing, even when SSH keys are in use. I have a lengthy, unintelligible password that I use for my UW account, so having to type this in any time I want to initiate a new SSH session on Mox is a painful process.

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Total Alkalinity Calculations - Yaamini’s Ocean Chemistry Samples

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I ran a subset of Yaamini’s ocean chemistry samples on our T5 Excellence titrator (Mettler Toledo) at the beginning of April. The subset were samples taken from the beginning, middle, and end of the experiment. The rationale for this was to assess whether or not total alkalinity (TA) varied across the experiment. If there was little variation, then there’d likely be no need to run all of the samples. However, should there be temporal differences, then all samples should be processed.

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FAIL - Missing Data on Owl!

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Uh oh. There appears to be some data that’s been removed from Owl. I noticed this earlier when trying to look at some of Sean’s data. His data should be in a folder with his name in Owl/scaphapoda

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RNA Isolation - Olympia oyster gonad tissue in paraffin histology blocks

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My previous go at this was a little premature - I didn’t wait for Laura to fully annotate her slides/blocks. Little did I know, the tissue was mostly visceral mass and, as such, I didn’t hit much in the way of actual gonad tissue. So, I’m redoing this, now that Grace has gone through and annotated the blocks to point out gonad tissue. SN-10-16 was sent to Katherine Silliman on 20170720.

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RNA Isolation - Olympia oyster gonad tissue in paraffin histology blocks

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UPDATE 20170712: The RNA I isolated below is from incorrect regions of tissue. I misunderstood exactly what this tissue was, and admittedly, jumped the gun. The tissue is actually collected from the visceral mass - which contains gonad (a small amount) and digestive gland (a large amount). The RNA isolated below will be stored in one of the Shellfish RNA boxes and I will isolate RNA from the correct regions indicated by Grace

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Data Received – Jay’s Coral epiRADseq - Not Demultiplexed

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Previously downloaded Jay’s epiRADseq data that was provided by the Genomic Sequencing Laboratory at UC-Berkeley. It was provided already demultiplexed (which is very nice of them!). To be completionists on our end, we requested the non-demultiplexed data set.

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Oyster Sampling - Olympia Oyster OA Populations at Manchester

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I helped Katherine Silliman with her oyster sampling today from her ocean acidification experiment with Olympia oysters (Ostrea lurida) at the Kenneth K. Chew Center for Shellfish Research & Restoration, which is housed at the NOAA Northwest Fisheries Science Center at Manchester in a partnership with the [Puget Sound Restoration Fund (PSRF)(http://www.restorationfund.org/). We sampled the following tissues and stored in 1mL RNAlater:

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Computing - Amazon EC2 Cost “Analysis”

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I recently moved some computing jobs over to Amazon’s Elastic Cloud Computing (EC2) in attempt to avoid some odd computing issues/errors I kept encountering on our lab computers (Apple Xserve 3,1).

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Dissection - Frozen Geoduck & Pacific Oyster

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We’re working on a project with Washington Department of Natural Resources’ (DNR) Micah Horwith to identify potential proteomic biomarkers in geoduck (Panopea generosa) and  Pacific oyster (Crassostrea gigas). One aspect of the project is how to best conduct sampling of juvenile geoduck (Panopea generosa) and Pacific oyster (Crassostrea gigas) to minimize changes in the proteome of ctenidia tissue during sampling. Generally, live animals are shucked, tissue dissected, and then the tissue is “snap” frozen. However, Micah’s crew will be collecting animals from wild sites around Puget Sound and, because of the remote locations and the means of collection, will have limited tools and time to perform this type of sampling. Time is a significant component that will have great impact on proteomic status in each individual.

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Opticon2 Calibration

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Jake and Steven recently noticed localized “hot spots” on most of Jake’s recent qPCRs, where higher levels of fluorescence were consistently showing up in interior portions of the plates than the outer portion of the plates.

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Reverse Transcription - O.lurida DNased RNA Controls and 1hr Heat Shock

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Performed reverse transcription on the Olympia oyster DNased RNA from the control samples and the 1hr heat shock samples of Jake’s project. To accommodate the large numbers of anticipated genes to be targeted in subsequent qPCRs, I prepared 100μL reactions (normally, 25μL reactions are prepared) using 250ng of each DNased RNA. A 1:10 dilution of the oligo dT primers (Promega) was prepared to improve pipetting accuracy. All incubations were performed in a thermal cycler without using a heated lid.

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Gel Purification - Olympia Oyster and Sea Pen PCRs

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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.

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DNA Isolation - Test Sample

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Due to the recent poor quality gDNA that has been isolated from C.gigas gonad, I decided to do a quick test using TE for DNA pellet resuspension in hopes that old Buffer EB (Qiagen) or old nuclease-free H2O (Promega) are to blame for the apparent, rapid degradation that I’ve experienced.

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DNA Quality Check - Yanouk’s Oyster gDNA

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We’ve had some Illumina sequencing issues with Yanouk’s samples, so I ran the samples out on a 0.8% agarose gel to evaluate the levels of degradation. Loaded 2uL of each sample. Did not load equal quantities of gDNA, due to the lack of available gDNA in the samples we submitted for Illumina sequencing. Added 2uL of H2O to samples 37 & 38 in hopes of having sufficient DNA for visualization on the gel.

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DNA Isolation - Geoduck

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Isolated additional geoduck gDNA from the two fresh (now frozen) geoduck’s that Brent provided me with on 20140212 so that we can potentially isolate RNA from the same geoducks to tie in with the DNA Illumina sequencing that Axa will be conducting. Isolated DNA using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocol (incubated minced siphon tissue at 56C for 3hrs). Eluted with 75uL of ddH2O and spec’d on NanoDrop1000.

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DNA Precipitation - Geoduck DNA from 20140213

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After speaking with Axa regarding the DNA concentrations, he would like the DNA from the ethanol-fixed tissue to be more concentrated, and he wants them in ddH2O instead of Buffer AE (from the Qiagen DNeasy Kit). So, I preformed a standard ethanol precipitation. Added 0.1 volumes of 3M sodium acetate (pH = 5.2) [15uL], 2.5 volumes of 100% ethanol [412.5uL] and incubated @ -20C over the weekend.

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DNA Isolation - Geoduck

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Since yesterday’s DNA isolation failed to yield sufficient quantity of DNA from the ethanol-fixed samples, I isolated additional DNA from the same samples.

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DNA Isolation - Geoduck

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Isolated gDNA from 6 geoduck siphon samples provided by Brent using the Qiagen DNeasy Spin Kit. Samples were as follows:

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Gylcogen and Carboyhydrate Assays - Emma’s C.gigas Whole Body Samples

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Samples were previously freeze dried overnight and stored @ -20C. To maximize sample homogeneity and, thus, increase accuracy of both assays, all samples were mechanically pulverized in their existing tubes. Approximately half of each sample was weighed and used for the glycogen assay. The remainder of each sample was stored @ -20C.

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PCR - Lake Trout C1q

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Repeated PCR from 20130919, but with a newly designed reverse primer. Primers were designed using MethPrimer (SR IDs: 1553, 1555). Primers were designed using MethPrimer:

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PCR - Lake Trout C1q

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Repeated PCR from 20130919, but with a newly designed set of primers that targets the desired region of C1q for sequencing (SR IDs: 1553, 1554). Primers were designed in Geneious.

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PCR - Lake Trout C1q

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Ran PCR on lake trout DNA and lake trout bisulfite-converted DNA. Used primers SRIDs: 1551 and 1552. DNA was isolated by Caroline Storer on 4/4/2011 and bisulfite converted on 4/7/2011. Master mix and cycling params are here:

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PCR - Hexokinase Partial CDS

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Performed PCR using the primers CG_HK_CDS_2132-2158 (SRID: 1521) and Cg_Hk_CDS_3’_no_stop (SRID: 1519) on pooled C.gigas cDNA (from DATE).

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PCR - Hexokinase Promoter and CDS

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Performed PCR to amplify the C.gigas hexokinase (ACCESSION#) promoter region (-2059bp) and the CDS without the stop codon. Elimination of the stop codon allows for subsequent cloning into the pBAD-TOPO expression vector, which will incorporate the V5 epitope tag sequence. This tag will be used to distinguish between endogenous hexokinase expression and expression generated from our hexokinase construct.

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Reverse Transcription - Herring RNA from 20091026

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Performed an RT reaction on pooled herring gonad and liver mRNA from 20091026 for James Raymond at the UNLV. A single RT reaction was performed using 12.75uL (208ng) of the pooled gonad mRNA and 5uL (132.5ng) of the pooled liver RNA, according to our default MMLV (Promega) protocol. After reaction was completed, sample was stored @ -20C and then shipped to James Raymond on 20130214.

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Reverse Transcription - FISH441 RNA

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Reverse transcribed the class FISH441 RNA to cDNA. Followed protocol provided on the FISH441 Wiki page, NOT the usual Roberts Lab protocol, with some modifications.

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Minipreps - Emma’s Illumina Library Cloning

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Performed plasmid isolation on 17 bacterial cultures Emma inoculated yesterday using the QIAprep Spin Mini Kit (Qiagen) using ~1.4mL of culture according to the manufacturer’s protocol. Plasmid DNA was eluted with 50uL of Buffer EB. Tubes were stored @ 4C in the refrigerator in FTR 213.

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Oligo Reconstitution - Illumina RNAseq Library Oligos and Barcodes

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Reconstituted all of the oligos and barcodes for library construction in TE (pH = 8.0) to a final concentration of 100uM. Created 10uM working stocks of all oligos and barcodes. All samples (stocks and working stocks) are stored @ -80C in their own box (Illumina Library Oligos & Barcodes) due to the fact that one of the oligos is an RNA oligo and requires storage at -80C.

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Chloroform Clean Up - Lexie’s QPX RNA from 20110504

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After submission of QPX samples to HTGU for Illumina library prep yesterday, I was notified that there was insufficient RNA for the QPX RNA samples. I checked the source RNA on the Roberts Lab NanoDrop1000 and determined that they had high phenol contamination (large peak at 270nm), which results in a large exaggeration in the OD260 absorbance (NanoDrop1000 report[JPEG]; notice terrible OD260/280 ratios; did not save screen shot of absorbance peaks.). As such, the concentrations that Lexie had listed in her notebook for these samples are highly inaccurate and highly inflated. To remove the phenol, I brought all of her QPX RNA samples from 20110504 up to ~200uL with 0.1%DEPC-H2O, added 200uL of chloroform, vortexed for 30s, spun at 12,500g RT for 15mins, and transferred aqueous phase to new tube. Then performed an ethanol precipitation on the aqueous phase. Added 0.1 vols of 3.0M sodium acetate (pH = 5.2), 2.5 vols of 100% EtOH, mixed and incubated at -20C for 1hr. Pelleted RNA by spinning at 16,000g 4C for 15mins.

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Reverse Transcription - DNased C.gigas Larval RNA from 20120427

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Performed reverse transcription using random primers (Promega) diluted 1:100 (5ng/uL) with 175ng of DNased total RNA. Random primers were used because we will be targeting V.tubiashii RNA instead of eukaryotic RNA. Reverse transcription was performed with M-MLV Reverse Transcriptase (Promega) according to the manufacturer’s protocol. Calcs are here.

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qPCR - Check DNased RNA from Earlier Today for Residual gDNA

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Ran qPCR using V.tubiashii VtpA primers (from Elene; no SR ID). Used 0.5uL of each DNased RNA sample, which equals ~40ng of RNA, which would be the equivalent amount of RNA that would end up in a qPCR rxn after cDNA has been made (using 1uL of cDNA). Used the filter DNA extraction from samples #279 from DATE as a positive control. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).

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gDNA Isolation - C.gigas Larvae from Taylor Summer 2011

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Samples that had been split from earlier today (see the RNA Isolation below) were resuspended in 1mL of DNAzol (MRC). 100ug of Proteinase K (Fermentas) was added to each sample. Samples were incubated at RT, O/N on a rotator. On 20120427 samples were pelleted 10mins, 10,000g, and supe transferred to fresh tube. DNA was precipitated with 0.5mL of 100% EtOH, mixed gently and pelleted 5mins, 5000g. Supe was discarded, pellets were washed with 1mL 75% EtOH, re-pelleted at same speed as previous step, supe discarded and pellets were resuspended in NanoPure H2O. Samples were spec’d on the Roberts Lab NanoDrop1000.

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RNA Isolation - C.gigas Larvae from Taylor Summer 2011

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Samples had been stored in RNA Later (Ambion). Samples were pelleted and the RNA Later supe removed. Samples were washed (2x) with 1mL TE (pH = 8.0) to remove excess salt resulting from the RNA Later. Samples were split, roughly equally, into two separate tubes. Samples were pelleted and the supe removed. One tube from each sample was set aside for gDNA isolation using DNAzol (MRC). The other tube was vortexed vigorously in TriReagent (MRC) and the then treated according to protocol. Samples were resuspended in 100uL of 0.1% DEPC-H2O and spec’d on the Roberts Lab NanoDrop 1000.

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qPCR - Taylor Water Filter DNA Extracts from 20120322

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Ran qPCR on the Taylor water filter DNA extracts from 20120322 using V.tubiashii VtpA primers (provide by Elene; no SR ID?) instead of 16s primers, which failed to produce acceptable results in the melt curves (see 20120323). Additionally, Elene has a standard curve for V. tubiashii (from 1/12/2011) based off of CFUs/mL, which will allow us to quantify theoretical number of V.tubiashii CFUs present in each sample.

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qPCR - Dave’s Manila Calm (Venerupis philippinarum) DNased RNA from yesterday and 20120302

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Performed qPCR on all DNased RNA samples from this group (samples #1-48) using beta actin primers (SR IDs: 1379, 1380). 0.5uL of each DNased RNA was used, which was the equivalent of ~40ng, in order to simulate the amount of RNA present in the subsequent cDNA (1000ng of RNA in 25uL cDNA; use 1uL of cDNA in qPCR reaction). Master mix calcs are here. Plate layout, cycling params, etc., can be found in the qPCR Report (see Results). 0.5uL of total RNA from sample Vp gill 01 was used to serve as a positive control, since Dave has no existing V. phillippinarum cDNA.

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qPCR - cDNA from 20120208

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Performed qPCR on all 12 samples. Used the following primers, provided by Elene, to detect V.tubiashii expression:

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qPCR - cDNA from earlier today

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Performed qPCR on all 12 samples. Used Cg_EF1aF/R2 (SR IDs: 1410 & 1412) for one set of qPCRs and Vtub_16s_F/R (SR IDs: 455 & 456) for the other set of qPCRs. Used pooled C.gigas cDNA (from 20110311) and RE22 DNA (provided by Elene) as positive controls for C.gigas and V.tubiashii, respectively. C.gigas gDNA (7ng of BB16 from 20110201) was used as a negative control for EF1a. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). All samples were run in duplicate.

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qPCR - DNased RNA from earlier today

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Checked DNased RNA samples from earlier today for the presence of residual gDNA. Used C.gigas BB16 gDNA (from 20110201) diluted to ~7ng/uL as a positive control to match the dilution factor of the RNA that will be used in the reverse transcription reaction (175ng in 25uL = 7ng/uL). All samples were run in duplicate. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

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RNA Isolation - C.gigas Larvae from 20110412 & 20110705 (Continued from 20120112)

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All of the RNA samples were re-combined with their respective counterparts and subject to a standard EtOH precipitation (0.1 volumes of 3M NaOAc, pH = 5.2, 2.5 volumes 100% EtOH; incubated -80C 1hr; pelleted; washed with 1mL 70% EtOH; pelleted). Pellets were washed two additional times (for a total of three washes) with 70% EtOH. RNA was resuspended in 50uL of 0.1% DEPC-H2O and spec’d on the Roberts Lab NanoDrop 1000.

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RNA Isolation - C.gigas Larvae from 20110412 & 20110705

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RNA was isolated from C.gigas larvae collected from Taylor Shellfish hatchery on the dates noted above. Samples were in RNA Later. RNA Later was removed. Attempted homogenization with a pestle proved futile, as a significant quantity of larvae were sticking to the pestle and were nearly impossible to wash off using TriReagent as a rinsing agent. Due to this, all samples were vortexed for 1min in 1mL of TriReagent. It should be noted that the TriReagent took on a cloudy appearance and even showed some separation into two layers upon letting the samples sit. This was not normal and I was immediately concerned about the high salt content from residual RNA Later. Samples were treated normally with the following changes:

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Mini-preps - COX/PGS Cloning Colonies from today

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Selected 10 colonies (1-8, 18, 28) for mini-preps. Inoculated 5mL 1x LB + 50ug/mL of Kanamycin. Incubated O/N, 37C, 200RPM. 3mL of each culture were used for mini-preps. Used Qiagen kit. Samples were eluted w/30uL of EB.

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PCR - COX/PGS Cloning Colony Screens from yesterday

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Performed PCR on 40 colonies using both qPCR primer sets to see if I could differentiate between which colonies potentially contained each isoform to reduce the amount of clones needed for sequencing. Master mix and cycling params are here. Primers used were:

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Cloning - Purified COX/PGS “qPCR Fragment” from 20111006

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Cloned the purified “qPCR Fragment” from 20111006 using the TOPO TA Cloning Kit (Invitrogen). Performed a half reaction (total volume = 3uL), using 1uL of purified PCR product. Incubated at RT for 20mins and then placed reaction on ice. Transformed chemically competent TOP 10 cells (Invitrogen) and heat shocked at 42C for 30s. Added 250uL of RT S.O.C. medium and incubated at 37C, 200RPM. Plated cells on pre-warmed Kan50+X-Gal plates (plates from 20110726; X-Gal added ~30mins before plating cells). Incubated plates O/N, 37C.

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Ethanol Precipitation - Full-length PGS1 cDNA (from 20110921)

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Performed an EtOH on gel-purified PCR products from 20110921. Briefly, added 0.1 vols of 3M sodium acetate (pH=5.2; 43uL), mixed and then added 2.5 vols of 100% EtOH (1182.5uL). Mixed, split into two tubes (due to high volume not fitting in a single tube) and incubated @ -80C O/N. Pelleted DNA 16,000g, 20mins, 4C. Discarded supe. Washed pellet w/ 1mL 70% EtOH. Pelleted DNA 16,000g, 15mins, 4C. Discarded supe. Resuspended both pellets in a TOTAL of 25uL Qiagen Buffer EB (10mM Tris-HCl) and spec’d.

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PCR - Full-length PGS1 cDNA

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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.

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Ethanol Precipitation - Purified PGS1 PCR from yesterday

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Added 0.1 vols of 3M sodium acetate (pH = 5.2; 38uL) and 2 vols of 100% EtOH (836uL). Incubated 30mins @ -20C. Pelleted DNA 16,000g, 15mins, 4C. Removed supe. Washed pellet w/1mL 70% EtOH. Pelleted DNA 16,000g, 15mins, 4C. Removed supe. Air-dried pellet. Resuspended in 50uL of Qiagen EB Buffer and spec’d.

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PCR - Full-length PGS2 cDNA

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Repeated PCR from 20110825 to attempt to amplify the full-length cDNA for PGS2 (COX2), however this time using a more robust polymerase (Amplitaq Gold) in hopes of getting results. Additionally, tried 3 different Mg2+ concentrations (1.5mM, 2.0mM, and 3.0mM). Master mix calcs and cycling params are here. cDNA was pooled cDNA made 20110311 from various tissues. PGS2 primers = 1376, 1375.

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Sequencing - C.gigas COX2/PGS2 Clone #4 from 20110728

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Used new primers for sequencing (SR IDs: 1351 & 1352) clone #4. Sequenced clone two times in each direction. DNA and primers were sent for sequencing at ASU. Requested “High GC” treatment to help overcome the issue seen on 20110728.

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Cloning - C.gigas COX2/PGS2 5’/3’ RACE Products (from earlier today)

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The bands that were excised and purified earlier today were cloned in to pCR2.1 using the TOPO TA Cloning Kit (Invitrogen) according to the manufacturer’s protocol with the following changes: used 4uL of all PCR products, incubated ligation reactions for 15mins @ RT, incubated competent cells with ligation reactions for 15mins on ice.

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5’/3’ RACE - C.gigas COX2/PGS2 Nested RACE PCR

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Performed nested RACE PCR on the RACE PCR products generated on 20110722 using the following nested primers: PGS2_ngspRACE_5’ (SR ID: 1350) and PGS2_ngspRACE_3’ (SR ID: 1349). Removed 2uL from each of the primary PCR reactions and brought up to 100uL in tricine EDTA (supplied in the Clontech SMARTer RACE cDNA Amplification Kit). Performed the nested RACE PCR according to the Clontech manual. Briefly, this is the same as the primary RACE PCR reaction, but using 5uL of the diluted primary PCR product and 1uL of the Nested Universal Primer (instead of 5uL of the 10X Universal Primer Mix). Master mix calcs and set up are here. Cycling params followed “Program 2” of the Clontech protocol, modified for nested primers, and are as follows:

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Clone Restreaking - PGS2 Hi/Lo Clones (from 20110421)

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Sequencing of the PGS2/COX2 clone failed (was empty vector). Restreaked bacterial clones on to a Kan50 plate (made 20110413 by SJW) from a plate that Caroline Storer had created from cloning colony selection on 20110421. Samples were labeled as PGS Lo 1 & 2 and PGS Hi 3 & 4. Additionally, there were red numbers on the plate associated with these four samples. They were 42 - 45, respectively. PGS Hi 4 (#45) was previously grown up and sequenced. This sample is what produced vector only sequence. Incubated O/N @ 37C. Hopefully we’ll bacteria is still viable and will have samples to grow up for miniprep, plamsid iso and sequencing.

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qPCR - C.gigas actin and GAPDH on V.vulnificus exposure cDNA (from 20110311)

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Ran a qPCR on all cDNA samples from the V.vulnificus exposure experiment from 20110111. This qPCR was to test 2 of 4 potential normalizing genes to evaluate which genes show the least amount of effect from the treatments in this experiment. Primers for actin used were Cg_Actin_306_F (SR ID: 1170), Cg_Actin_408_R (SR ID: 1171). Samples were run in duplicate. Master mix calcs are here. The master mix info is the same that was used earlier today, but with the primers noted above, not those listed on the calcs page. Plate layout, cycling params, etc., can be seen in the qPCR Report (see Results).

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qPCR - C.gigas 18s and EF1a on V.vulnificus exposure cDNA (from 20110311)

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Ran a qPCR on all cDNA samples from the V.vulnificus exposure experiment from 20110111. This qPCR was to test 2 of 4 potential normalizing genes to evaluate which genes show the least amount of effect from the treatments in this experiment. Primers for 18s used were Cg_18s_1644_F (SR ID: 1168), Cg_18s_1750_R (SR ID: 1169). Primers for EF1a used were EF1_qPCR_5’ (SR ID: 309), EF1_qPCR_3’ (SR ID: 310)Samples were run in duplicate. Master mix calcs are here. The master mix info is the same that was used earlier today, but with the primers noted above, not those listed on the calcs page. Plate layout, cycling params, etc., can be seen in the qPCR Report (see Results).

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qPCR - C.gigas COX2 on V.vulnificus exposure cDNA (from 20110311)

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Ran a qPCR on all cDNA samples from the V.vulnificus exposure experiment from 20110111. Primers used were Cg_COX1/2_qPCR_F (SR ID: 1192) & Cg_COX2_454align1_R (SR ID: 1190). Samples were run in duplicate. Master mix calcs are here. The master mix info is the same that was used earlier today, but with the primers noted above, not those listed on the calcs page. Plate layout, cycling params, etc., can be seen in the qPCR Report (see Results).

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qPCR - Hard Clam NGS Primer Checks

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Ran a qPCR to evaluate a large batch of primers (40 sets) that were ordered per Steven, based off of the most recent SOLiD run (samples submitted 3/10/2011; see Dave’s notebook for more info). Pooled cDNA (2uL from each individual; from 20110511) was used. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results). The list of primers tested is available in the Primer Database and consist of SR IDs 1233 - 1312. For brevity, samples were only labelled with the corresponding contig number.

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qPCR - Emma’s New 3KDSqPCR Primers

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Due to previous contamination issues with Emma’s primers, Emma asked me to order new primers, reconstitute them and run a qPCR for her to see if we could eliminate her contamination issues with this primer set. cDNA template was supplied by Emma (from 2/2/11) and was from a C.gigas 3hr Vibrio vulnificus challenge. Samples were run in duplicate, as requested. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results). Primer set used was:

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DNase - Hard Clam Gill RNA (from earlier today)

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DNased 5ug of RNA from each sample with Ambion’s Turbo DNA-free Kit, according to the manufacturer’s rigorous protocol. Samples were spec’d and stored @ -80C in the “Hard Clam V.t. Experiment RNA” box.

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Ligations - COX1/COX2 PCR Products

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Performed ligations/cloning on a variety of COX1 genomic and COX 5’ RACE products using the TOPO TA Cloning Kit (Invitrogen). Used 2uL of gel-purified PCR product in each cloning rxn, 2.5uL of H2O, 1uL of salt solution, and 0.5uL of the Invitrogen pCR2.1 vector. Incubated samples for 5mins at RT. Used 2uL of the cloning reaction to transform TOP10 chemically competent cells (Invitrogen), mixed very gently, incubated on ice for 5mins, heat shocked at 42C for 30s and immediately placed on ice. Added 250uL of SOC Medium and incubated tubes at 37C, 200RPM for 1hr. Plated 50uL of each transformation on LB+Kan plates (made by Steven on unknown date with unknown Kan concentration) containing 40uL of 40mg/mL X-gal. Incubated O/N at 37C. Remaining volume of transformed bacteria were stored @ 4C.

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5’/3’ RACE PCRs - Nested PCRs for COX2 Sequence

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Due to the failure of the primary PCR on both 5’ and 3’ RACE cDNA libraries (from 20080619) yesterday, will perform nested PCR using a nested GSP designed by Steven (CgPGSRACEsrNGSP1; SR ID:1209). The 5uL of PCR reactions that were set aside yesterday were diluted to 250uL with tricine-EDTA (supplied with the Clontech SMARTer RACE cDNA Amplification Kit) as instructed in the Clontech manual. The master mix and tube layouts were exactly the same as yesterday’s, but instead of using 2.5uL of RACE cDNA library as template, I used 5uL of the diluted PCR reaction. Additionally, Universal Nested Primers were used instead of the Universal Primer Mix (both supplied in the kit). Cycling parameters followed “Program 2” from the Clontech manual for 25 cycles.

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5’/3’ RACE PCRs - COX2 Sequence on 5’ & 3’ RACE Libraries (from 20080619)

  • ~1 min read

Ran PCRs on both the 5’ & 3’ RACE libraries created 20080619 with a new COX2 gene-specifc (GSP) primer designed by Steven (CgPGSRACEsrGSP1; SR ID: 1208). Although this primer was designed to obtain additional 5’ sequence, it was used with both 5’ and 3’ libraries as a precaution in case it accidentally designed on the wrong strand. PCR rxn was set up according to the Clontech SMARTer RACE cDNA Amplification Kit. Master mix calcs are here. PCR cycling followed “Program 1” from the Clontech manual for 25 cycles.

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mRNA Isolation - Pooled Black Abalone Dg RNA (from Abalone Dg Exp 1)

  • ~1 min read

mRNA was isolated for SOLiD sequencing by HTGU. Made two pools of San Nick RNA (Control and Exposed) using equal amounts (5ug) of each individual sample. Individual samples used can be found here. mRNA was isolated using Ambion’s Micro PolyAPurist Kit according to protocol. Procedure was performed two times on each pool and then EtOH precipitated. Samples were resuspended in 10uL of The RNA Storage Solution provided in the kit and spec’d on the Roberts Lab ND1000. Samples were stored @ -80C in the “Next Gen Sequencing Libraries” box.

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3’RACE - C.gigas 3’RACE for COX2

  • ~1 min read

Used Cg_COX2_3’RACE_short (SR ID: 1197) & Cg_COX2_3’RACE_long (SR ID: 1196) and the Clonetech SMART RACE cDNA Amplification Kit (unknown acquisition date) to attempt to acquire more 3’ sequence of the C.gigas COX2 isoform. Used Gigas 3’RACE cDNA (from 20080610).

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NanoDrop1000 Comparison - Roberts vs. Young Lab

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A previous comparison was performed (see 20110209), but it was determined that the standard DNA being used to test the machines was old/degraded. Lisa ordered a new standard DNA dilutions series (Quant-iT dsDNA Kit; Invitrogen) and these DNAs were used. All DNAs were measured 5 times and were mixed by gently flicking between each measurement. A “blank” was measured between each different [DNA] and, if the reading was > + or - 1ng/uL, the machine was reblanked.

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Data Analysis - Young Lab ABI 7300 Calibration Checks

  • 2 min read

All runs (3 runs were conducted) were created using a master mix containing C.gigas gDNA (either 50ng or 100ng), 1X Promega qPCR Master Mix, 0.2uM each of forward/reverse primers (18s; Roberts SR ID: 156, 157). The master mix was mixed well and 10uL were distributed in each well of ABI plates. Plates were sealed with ABI optical adhesive covers.

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NanoDrop1000 Comparison - Roberts vs. Young Lab

  • 2 min read

Due to an apparent reduction in assay sensitivity for the Hematodidium qPCR assay, we have decided to determine if the spec readings of the plasmid DNA being used for the standard curves are accurate. Used C.gigas gDNA and the lambda DNA Standard (100ng/uL) included in the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) that was marked as received 9/1/10. Tested both the Roberts Lab and Young Lab using these DNAs. At least 6 separate measurements were taken of each DNA on each machine. Samples were briefly mixed by flicking the tube 4-5 times prior to each measurement.

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qPCR - Test Young Lab qPCR Calibration (Repeat)

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This is a repeat of a run from 20110204. Here’re master mix calcs. This was being repeated to evaluate whether or not the relative differences in Ct values observed on 20110204 are consistent or not across the plate. Cycling params were as follows:

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qPCR - Test Young Lab qPCR Calibration (Repeat)

  • ~1 min read

This is a repeat of an earlier run from today, but with a different qPCR plate. [Here’re master mix calcs (bottom half of page)(https://eagle.fish.washington.edu/Arabidopsis/Notebook%20Workup%20Files/20110204-01.jpg). Cycling params were as follows:

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qPCR - Test Young Lab qPCR Calibration

  • ~1 min read

Recently, the Young Lab’s ABI 7300 qPCR machine was calibrated. Steven asked me to run a plate and see how well the calibration worked. Ran a plate with C.gigas gDNA and Gigas 18s primers (SR ID: 156 and 157) that are known to amplify gDNA. [Master mix calcs are here (top half of page)(https://eagle.fish.washington.edu/Arabidopsis/Notebook%20Workup%20Files/20110204-01.jpg). Cycling params were as follows:

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Gigas Bacterial Challenge - 1hr & 3hr Challenges with Vibrio vulnificus

  • ~1 min read

400mL O/N culture (1x LB+1% NaCl, 37C, 150RPM, 1L flask) of V.vulnificus (STRAIN??) and V.tubiashii (Strain: RE22) were pelleted (4300RPM, 25C, Sorvall ST-H750 rotor). Supe was removed and pellets were each resuspended in 50mL sea water. 1mL was taken from each to use for dilutions to determine colony forming units (CFU).

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