Tag Archives: larvae

DNA Isolation – C.gigas larvae from 2011 NOAA OA Experiment

DNA was isolated from the following samples:

SAMPLE ID DATE TREATMENT (ppm) # LARVAE
6B5 20110513 400 5,000
1B2 20110513 1000 5,000
6B2 20110513 400 10,000
1B1 20110513 1000 10,000
1B1 20110519 1000 NA
1B2 20110519 1000 NA
6B2 20110519 400 NA
6B1 20110519 400 NA

 

Some tubes contained a high quantity of algae, based on quantity of material in tube and overall green color.

Samples 1B1 & 1B2 from 20110519 have excessive quantities of algae.

Samples 6B1 & 6B1 from 20110519 have a fair amount of algae.

See pic:

 

Sample tubes after brief spin, prior to DNA isolation.

Prior to isolation, samples were briefly spun (12,000g, 15s @ RT). Supernatants were discarded.

 

 

DNA Isolation

DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen).

Samples were resuspended in 180uL of Buffer AL and 20uL of Proteinase K. Samples were mixed by vortexing and incubated @ 56C O/N.

The manufacturer’s protocol (Purification of Total DNA from Animal Tissues (Spin-Column Protocol)) was followed.

Due to low quantities of starting tissue, samples were eluted with 200μL of Buffer EB to maximize DNA recovery.

 

DNA Quantification

Samples were prepared for quantification via fluorescence using the Quant-iT DNA BR Kit (Life Technologies/Invitrogen). The manufacturer’s protocol was altered to use 5μL of sample and 5μL of standards (instead of 10μL) in each well. All samples/standards were run in duplicate and read on a FLx800 plate reader (BioTek).

Mean fluorescence of the standards were plotted with a best-fit line. The resulting equation from the best-fit line was used to determine sample concentrations from their mean fluorescence.

 

Results:

Calcs and resulting quantities are here:

https://docs.google.com/spreadsheets/d/1e7EF05akWeBtO7Xz0UWXhIzRlkVU5HA_rjCE3c4SPEw/edit?usp=sharing

 

All samples have yields great enough to proceed with shearing and bisulfite conversion.

Samples 1B1 and 1B2 from 20110519 have extremely large yields.  This is not surprising, considering the amount of algae present in the source tubes.  Will process only 500ng from each sample.

 

 

DNA Shearing

Adjusted volume of all samples to 190μL using Buffer EB (Qiagen) in 1.5mL snap-cap tubes.

Samples were sonicated/sheared in the Bioruptor (Diagenode) with the following cycling protocol:

25 cycles of:

30s on

30s off

Cycling params were adjusted from the last time I performed this, since I felt the final sheared size was a bit on the small size.

After shearing, samples were stored @ 4C until I could SpeedVac them to reduce their volumes, as the bisulfite treatment step requires volumes < 24uL.

 

DNA Isolation – C.gigas Larvae from Emma OA Experiments

Isolated gDNA using DNazol from the following larval samples for potential MBD selection and bisulfite sequencing:

– Added 500uL of DNAzol to each tube, transferred to 1.5mL tube and homogenized with disposable pestles

– Added additional 500uL DNAzol to each homogenized sample and mixed by inversion.

– Incubated 10mins at RT

– Pelleted debris by spinning 10,000g, 10mins, @ RT

– Transferred supes to new tubes

– Added 500uL of 100% EtOH to each; mixed by inversion; incubated 10mins @ RT

– Pelleted DNA by spinning 5,000g, 4mins, @ RT

– Discarded supes

– Washed DNA with 1mL 70% DNAzol/30% EtOH solution

– Spun 1000g, 1min, @ RT

– Discard supes

– Washed DNA with 1mL 75% EtOH

– Spun 1000g, 1min, @ RT

– Discarded supes

– Spun 1000g, 1min, @ RT

– Removed residual EtOH with pipette; air dried samples for 5mins @ RT

– Added 20uL of Trish-HCl (pH = 8.0) to each sample; incubated 10mins @ RT; flicked tubes to help dissolve

– Spun 12,000g, 10mins, @ RT

– Transferred supes to new tubes

– Spec’d on NanoDrop 1000 (ThermoFisher) and recovered solution from each sample due to limited sample volume

Results:

DNA Isolation – C.gigas Larvae from Katie Latterhos and Emma

Isolated gDNA from two C.gigas larvae samples (stored in RNA Later) from Katie Latterhos:

B4 400 D05

B6 400 D03

and two samples from Emma:

280E

380E

Emma’s samples were in her -80C box (in rack #2): C.gigas larvae – NOAA O.A. September 2010 Emma

Note: No visible larvae present in either of Katie Latterhos samples. Easily visible larvae in each of Emma’s samples.

DNA was isolated using the Quick-gDNA MicroPrep Kit (ZymoResearch) according to the manufacturer’s protocol for Cell Suspensions and Proteinase K Digested Samples.

Samples were eluted with 10uL of elution buffer and spec’d on a NanoDrop1000 (ThermoFisher). The 2uL used for each sample were recovered from the NanoDrop.

Results:

Not surprisingly, the samples from Katie Latterhos yielded, essentially, no gDNA. Will discuss with Steven.

*UPDATE 20141030*

Steven sent me this screen cap of Emma’s notebook so we could track where the samples originated from

qPCR – Manila Clam Larvae cDNA (from August 2012 – Dave’s Notebook)

Ran qPCR on manila clam larvae cDNA that Dave made on 8/7/2012, using the sample sets from 7/29/2011 and 8/5/2011 of the OA manila clam experiment he ran.

Primers used:

Rp_GPX3_F/R2 (SR IDs: 1453, 1469)

Rp_HSP90_F2/R2 (SR Ids: 1457, 1471)

Primers were verified to be in good working order by Dave on 4/1/2012 (see Dave’s notebook).

Master mix calcs are here. Cycling params can be found in the qPCR Data File (see Results). Plate layout and PCR Miner analysis can be found in the qPCR Raw Data file (see Results). All samples run in duplicate.

Results:

qPCR Data File(Opticon 2) http://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20121108_150936.tad

qPCR Raw Data and PCR Miner Analysis(Excel) http://eagle.fish.washington.edu/Arabidopsis/qPCR/Opticon/Sam_20121108_150936.xlsx

Reps look pretty good, although the 4C2 8.5.11 sample has consistently bad reps across all of today’s runs. Data will be shared with Steven for comparison to Dave’s Illumina data.

All data was normalized to EF1a expression from later today.

qPCR – DNased Manila Clam Larvae RNA (from August 2012 – Dave’s Notebook)

Performed qPCR on Dave’s manila clam larvae DNased RNA from August 2012 using EF1a primers (SR IDs: 1463, 1474).

Master mix calcs are here. https://docs.google.com/spreadsheet/ccc?key=0AmS_90rPaQMzdHc5amwzZzdDa1d0VXQzLVU0WkFTc0E

Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Positive control was pooled cDNA taken from Dave’s cDNA plate on 8/7/2012.

Results:

qPCR Data File(CFX96) http://eagle.fish.washington.edu/Arabidopsis/qPCR/CFX96/Roberts%20Lab_2012-10-26%2010-48-07_CC009827.pcrd

qPCR Report(PDF) http://eagle.fish.washington.edu/Arabidopsis/qPCR/CFX96/Roberts%20Lab_2012-10-26%2010-48-07_CC009827.pdf

Here’s a quick Google Spreadsheet summary highlighting samples that came up positive/negative.

https://docs.google.com/spreadsheet/ccc?key=0AmS_90rPaQMzdFFHb3YwWE01UG00TnY3OWo2cWx2UVE

Approximately half of the samples (~27) came up positive for still having gDNA in them.

There are three pCO2 treatments: 1000ppm, 750ppm, and 400ppm. There are six sampling dates: 7/29/2011, 8/2/2011, 8/9/2011, 8/12/2011. Currently, it is unknown when the Day 0 samples were collected. Have emailed Dave for deets.

There are only two dates (7/29/2011 and 8/5/2011) that have a full set of samples (i.e. 1000ppm, 750ppm and 400ppm) that exhibit DNA-free RNA. Will discuss with Steven on how to proceed.

UPDATE 20121031 – Dave emailed and indicated the experimented started on 7/27/2011. Additionally, the two sample sets that are complete are Day 2 and Day 7. Discussing with Steven, we have decided to run a few genes and see how the expression levels compare to the NGS data analysis for these samples. If the qPCR data supports the NGS data, then that information will be relayed to the BMC Genomics reviewers in response to their critiques. A copy of the manuscript is here(may not be publicly viewable). https://docs.google.com/document/d/1Ii1lODz2oThiyxZtHBblUEdzyhIVq92n8jkEjhkuuts/edit

qPCR – Detection of V.tubiashii Presence and Expression Using VtpA Primers in DNA/cDNA from yesterday

Ran qPCR with VtpA primers on cDNA and DNA (from yesterday) of C.gigas larvae to see levels of V.tubiashii compared to their water filter samples (see 20120326). Master mix calcs are here. Plate layout, cycling params, etc can be seen in the qPCR Report (see Results). Used 1uL of cDNA and 100ng (1uL) of DNA as template.

All samples were run in duplicate.

Results:

qPCR Data File (CFX96)
qPCR Report (PDF)

No detectable levels of expression (or, no expression at all) in any of the cDNA samples.

Below I’ve put together a very rough comparison of larvae levels, based off of the the standard curve. I have NOT done the full back calculations!! This is data straight out of the qPCR machine, using the standard curve. Due to the large range, I’ve graphed the data on a logarithmic scale so all the data is visible on the graph.

qPCR – Check DNased RNA from Earlier Today for Residual gDNA

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).

Results:

qPCR Data File (CFX96)

qPCR Report (PDF)

All samples showed up negative, except for the positive control. Will proceed with making cDNA on Monday.

gDNA Isolation – C.gigas Larvae from Taylor Summer 2011

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.

Results:

Report on the NanoDrop software wouldn’t display, so I’ve entered the concentration of each sample in the table below.

SampleID ng/uL
201 1364
280 131.7
314 710.3
342 539.4
434 274.4
552 334.8
605 369.7

 

RNA Isolation – C.gigas Larvae from Taylor Summer 2011

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.

Results:

Overall, the samples look really good. Some samples (280, 434 & 605) required re-specing after the NanoDrop was reblanked in order to get a reading without an error message. They will be DNased and then reverse transcribed.

RNA Isolation – C.gigas Larvae from 20110412 & 20110705

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:

  • Aqueous phase after chloroform treatment was clear, but grey in color. This is not necessarily unusual.

  • Addition of isopropanol triggered immediate precipitation of a dark grey material.

  • “Pelleting” of the RNA after the isopropanol precipitation resulted in a gooey grey material that did NOT pellet, and a clear supernatant. The grey goo was transferred to a clean tube. An additional 500uL of isopropanol was added to the clear supernatant of two samples (#140 & #142), as well as to the grey goo. The addition of isopropanol to the clear supe resulted in an immediate precipitation of white salt-like material. The isopropanol appeared to have no effect on the grey goo. All samples were stored @-20C in their existing conditions until 20120116.

  • Since the two samples that were treated with an additional 500uL of isopropanol produced an excess of salt precipitation, I instead added 1mL of 70% EtOH to all the remaining samples; both the clear supernatants and the grey goo. The idea being that the higher water content in the 70% EtOH would help to keep the salts in solution, while precipitating the RNA. Samples were pelleted. All of the grey goo samples produced a white pellet. The grey goo seemed unchanged. Supernatants (including grey goos) were discarded and the resulting pellets from all samples were washed in this fashion were washed three more times.

  • Pellets were resuspended in 25uL of 0.1% DEPC-H2O and stored @ -80C until 20120123.

  • Samples were spec’d on the Roberts’ Lab NanoDrop 1000.

Results:

Spreadsheet of OD readings is here.

Since samples were split into two (clear supernatant and grey goo), they were kept separate through the remainder of the process. Sample names are appended with “-1″ or “-2″. “-1″ samples are grey goo samples and the “-2″ samples are the clear supernatant samples.

Overall, most of the grey goo samples appear to have produced the highest yields and highest quality of RNA, although this is not true for all of the samples.