20090526

gDNA Isolation - C. pugetti (from 20090518)

Followed JGI "Bacterial genomic DNA isolation using CTAB" protocol(Word doc) with the following notes/changes.
Two, 1L cultures were transferred to 4 total jars (~500mL in each jar) for centrifugation. Jars had been briefly washed with 95% EtOH prior to use.
Cells were pelleted in Sorval T21 using a bucket rotor for 30mins., 4200RPM, 25C.
Supe was removed and all four pellets were resuspended with a total of 10mL of TE.

OD600 = ~2.1, so added an additional 10mL of TE.
OD600 = ~1.2, so proceeded with procedure.

Due to volume of the prep, the sample was split into two, 50mL centrifuge tubes (washed with 95% EtOH prior to use) prior to the addition of 5M NaCl.

Disaster strikes! Turns out the tubes being used were not resistant to chloroform. This was realized after spinning at 18,000RPM, 25C 10mins in a Sorval SL-50T rotor in the Sorval T21 centrifuge.

However...

I recovered the aqueous phase anyway, as it was still contained in the tube and had no contact with the rotor surface(s). Due to a subsequent phenol:chloroform:IAA step, I split the aqueous phase into 24 x 1.5mL snap cap tubes and proceeded according to protocol. The final DNA pellet, prior to resuspension were combined from all the tubes into a single 1.5mL snap cap tube in a final volume of 400uL of TE.


external image 20090528%20gDNA%20SJW.bmp

Results: DNA from today looks good with excellent yield. DNA from 20090513 doesn't look as nice AND the concentration doesn't jive with the QC gel that was run on 20090513. Will run out on gel according to JGI protocol to evaluate quality further.


20090519

DNA Methylation Test - Gigas site gDNA (BB & DH) from 20090515

Used BB & DH samples #11-17 for procedure. Followed Epigentek's protocol. My calcs for dilutions/solutions needed are here. All solutions were made fresh before using, except for Diluted GU1 which was made at the beginning of the procedure and stored on ice in a 50mL conical wrapped in aluminum foil. Used 100ng total (50ng/uL) of each sample gDNA. No standards for a standard curve based on speaking with Mac.

WELL
SAMPLE
WELL
SAMPLE
A01
BB11
A02
DH11
B01
BB12
B02
DH12
C01
BB13
C02
DH13
D01
BB14
D02
DH14
E01
BB15
E02
DH15
F01
BB16
F02
DH16
G01
BB17
G02
DH17
H01
Pos. Control
H02
Blank
external image 20090519%20gigas%20methylation%20BB%20vs%20DH%20graph.jpg

Results: Above is the graph of the results. Although it's only a small difference between the two sites, it is statistically significant. The calcs for this graph can be found here (Excel file). It should be noted that this graph was generated using estimated values from the standard curve provided in the manufacturer's protocol. This was done because 1) I did not run standards to generate my own curve and 2) calculating the "% methylation" not using the formula that utilizes the standard curve was giving ridiculously high values (e.g. 350%).

Here is the raw data generated by the plate reader for a 1s read (Excel file) and a 0.1s (Excel file) read. Both reads have nearly identical values.


20090518

RNA Isolation - Abalone Dg Project samples

Isolated RNA from Abalone Dg samples (see below) using MoBio RNA PowerSoil Kit according to protocol. RNA was stored @ -80C.

external image 20090519%20RNA%20SJW-01.bmp

Results: RNA quality looks great, as usual. Sample 06:6-44 has a very low yield, but was to be expected due to very small amount of starting tissue.

C.pugetti - Liquid Cultures

Inoculated 2 x 1L of 1x Marine Broth + biphenyl crystals (in 2L flasks) using 5mL of liquid culture from 20090501. Incubated at 28C 200RPM.


20090515

gDNA Isolation - Gigas Dermo Samples

Transferred tissue from previously Chelexed samples:
WP3, WP7, WP13, WP14, WP15, CY1 - These all tested negative for Virginica gDNA (18s qPCR by Rony on DATE)
CY42, CY36 - These tested positive for Virginica gDNA (18s qPCR by Rony on DATE).
Used Qiagen DNeasy Kit. Digested samples for 3 hrs. at 55C in Proteinase K according to protocol.

external image 20090515%20gDNA%20Dermo%20SJW-01.bmp

Results: Horrible. Virtually no DNA in any of these samples. Additionally, the quality of the DNA is atrocious. Not sure what to do now.

gDNA Isolation - Mac's BB and DH site samples

Due to failure of gDNA isolation via the TriReagent method (see 20090511) used Qiagen DNeasy Kit. Digested samples for 3 hrs. at 55C in Proteinase K according to protocol. Performed on a subset of each site samples: BB#11-18 & DH#11-18.

external image 20090515%20gDNA%20SJW-01.bmp

Results: Excellent yields and superb quality.


20090514

Reverse Transcription - Mac's gigas DNased RNA from 20090512

Performed RT using Promega M-MLV RT according to M-MLV protocol and used 0.5ug oligo dT per ug of RNA on all BB and DH site samples that were negative for gDNA (see qPCR results 20090512). Calculations and work up are here. Samples were set up in a plate to facilitate sample loading in subsequent qPCRs.

UPDATE: cDNA plate was discarded 20120320 by SJW.


20090513

gDNA Isolation - C.pugetti


Isolated according to JGI protocol (Word doc). Used 100mL, 8 day old culture inoculated from a plate on 20090505. Resuspended pellets in 740uL of TE and took an OD600 via the NanoDrop. Diluted the sample appropriately to an OD600 ~ = 1.0 in a final volume of 740uL TE (see the last three measurements for OD600 of final dilution).

Followed protocol. Recovered 300uL of aqueous phase prior to precipitation with isopropanol (Step #21). Resuspended DNA in 20uL of H2O. Will run samples on gel according to JGI instructions.

external image 20090514%20C%20pugetti%20gDNA%20JGI%20QC.jpg

Lane 1 - 15ng standard (5uL)
Lane 2 - 31ng standard (5uL)
Lane 3 - 63ng standard (5uL)
Lane 4 - Marker 2 (5uL)
Lane 5 - C. pugetti DNA (5uL: 3uL + 2uL dye)
Lane 6 - Marker 3 (5uL)
Lane 7 - 125ng standard
Lane 8 - 250ng standard (5uL)
Lane 9 - 500ng standard (5uL)

Results: DNA looks stellar! Just like the example gel in the JGI QC documentation. however, looks to be too little TOTAL yield of DNA to send for sequencing (need


20090512

qPCR - Mac's gigas DNased RNA from earlier today

Performed qPCR on the DNased oyster RNA from earlier today with Gigas_18s_F/R primers to verify removal of detectable gDNA in the samples, since the qPCR from 20090508 indicated residual gDNA was still present in some of the DNase treated RNA. Plate layout/set up is here.

Results: Samples BB# 2, 5, 6, 15 still came up as positive for gDNA conatimntion. These will NOT be used to make cDNA for subsequent qPCRs.

DNase Treatment (Rigorous!) - Mac's gigas RNA/Re-DNased RNA from 20090507 & 20090508, respectively

Followed the rigorous protocol for Ambion's Turbo DNA-free protocol for the following RNAs:

BB#11-20
DH#11-20

Used 10ug of RNA (200ng/uL) in 50uL as directed. Here are the calcs for FF and DH.

Followed standard protocol on DNased samples from 20090508:

BB#1-10
DH#1-10

The standard protocol should be fine for these samples, since the procedure worked on Friday for some of them.




20090511

DNA Isolation - Mac's gigas samples from 20090505 & 20090506

Isolated gDNA according to MoBio TriReagent protocol from BB#1-20 and DH#1-20. Resuspended DNA in 600uL of 8mM NaOH. Spec.

external image 20090512%20gDNA%20plots%20SJW.jpg


Results: HORRIBLE! This is some of the worst "DNA" I've ever seen. Peaks everywhere EXCEPT at 260nm. Here's a link to the actual numbers. It's a text file and is comma separated, so you should open with Excel for it to be readable.

Spoke with Steven. Will pursue RNA instead of continuing down this path for now.




20090508

qPCR - Re-DNased oyster RNA from today

Performed qPCR on the re-DNased oyster RNA from earlier today with Gigas_18s_F/R primers to verify removal of detectable gDNA in the samples, since the initial qPCR from yesterday indicated residual gDNA was still present in the DNase treated RNA. Plate layout/set up can be found here.

Results: About 4 samples in each site set are NEGATIVE for gDNA. That means the remainder still have detectable levels of gDNA. Boo.

DNase Treatment - Oyster RNA from yesterday

Yesterday's qPCR indicated that all of the RNA still contained gDNA contamination. So, took 10ug of RNA from BB #1-10 and DH#1-10 (calcs/workup BB and DH) and brought the volumes up to 50uL with 0.1% DEPC-H2O. Processed the samples according to Ambion Tubrbo DNA-free protocol. Will proceed with qPCR immediately.



20090507

qPCR - DNased oyster RNA from earlier today

Performed qPCR on the DNased RNA to with Gigas_18s_F/R primers to verify removal of detectable gDNA in the samples. Plate layout/set up can be found here.

Results: All samples produced a signal. In retrospect, this is likely due to having too much RNA for the DNase treatment. I proceeded with the DNase treatment and qPCR prior to specing the samples. Spec revealed that most of them were highly concentrated; more than the Ambion protocol recommends. Will redo the DNase treatment on a subset of the samples using the appropriate quantity of RNA.

DNase Treatment - Oyster RNA from today

All of the RNA (50uL) was DNase treated with Ambion's Turbo DNA-free kit according to protocol. Samples were spec'd.

Results:

external image 20090507%20RNA%20SJW-01.jpg

external image 20090507%20RNA%20SJW-02.jpg


RNA Isolation - Mac's oyster tissues (BB and DH) (CONTINUED from yesterday)

Samples were precipitated according to TriReaget protocol. RNA was resupended in 50uL of 0.1% DEPC-H2O and then heated @ 55C for 10mins to help dissolve the pellets. Samples will be DNase treated.


20090506

RNA Isolation - Mac's oyster tissues (BB and DH) (CONTINUED from yesterday)

Completed the reaminder of the samples (BB#9-20 and DH#1-20) up to the point of precipitation. Isopropanol was added and stored @ -20C. Organic phase was retained for subsequenct gDNA isolation and stored @ 4C.


20090505

Bacteria - C. pugetti liquid cultures


Started two 100mL cultures in 1x Marine Broth + biphenyl. One culture was inoculated from the original liquid culture (from 20090419) and the second was inoculated with a thick loopfull of C. pugetti from the plate (from 20090424). Cultures were incubated @ 28C, 200RPM.

RNA Isolation - Mac's oyster tissues (BB and DH)

Processed BB#1-8 up to the point of precipitation. Added isopropanol and stored @ -20C. Organic phase was retained for subsequent gDNA isolation and stored @ 4C.


20090501

Bacteria - C. pugetti liquid cultures

Inoculated a total of 10, 5mL 1x Marine Broth in 50mL conicals. 5 tubes received 1mL of the original culture started on 20090419. 4 tubes received 1mL of the secondary culture (from 20090421). 1 tube was inoculated with a colony from the plate streaked on 20090424. Incubated all tubes @ 28C, 200RPM. Used a higher temp. to encourage faster/more robust growth.


20090430

Bacteria - C. pugetti plate (from 20090424)

An additional yellowing has occurred on the plate. This is in accordance with the Dyksterhouse et al. paper. Colonies still barely visible.


20090429

Bacteria - C. pugetti plate (from 20090424)

There appears to be growth on the plate. There is a large, bright yellow/chartreuse region on the plate where the streaking was initiated. Additionally, upon close inspection, it appears as though colonies can be seen.

Oyster pH - Tank set up

Made sea water using Instant Ocean according to recipe on the bucket (1/2 cup per gallon).



20090427

Bacteria - C. pugetti liquid culture


Results: It has now been 8 days since revival of the ATCC freeze dried culture. The media still looks a bit cloudy, but hasn't really changed since the second or third day post-revival. Of note, there does seem to be an accumulation of clumps in the tube. These may or may not be clumps of cells. Will consult with Steven when he returns. Might need to pass cells and take some ODs to really assess changes in growth as these cells may not be as robust as E. coli or other common lab cultures.


20090424

Bacteria - C. pugetti plate

Streaked C. pugetti onto marine broth plate + a few biphenyl crystals and incubated at RT over the weekend.

Results: No growth as of Monday, 20090427.


20090422

Reverse Transcription - cDNA from DNased Abalone RNA from 20090420


Prepared cDNA using an equal amount of RNA from all samples (442.6ng). This amount was based on using the maximum allowable volume of RNA for the RT rxn AND the sample with the lowest [RNA] (08:3-20; 24.59ng/uL). cDNA was prepared according to the Promega MMLV RT recommendations. Here is the work up for the cDNA rxns. cDNA was stored @ -20C.



20090421

qPCR - Abalone DNased RNA from yesterday

Performed qPCR to evaluate gDNA removal w/ 2x Immomx and SYTO 13. qPCR/plate set up is here.

Results: The two cDNA samples come up as positive. No flourescence detected in any other gamples. However, melting curves look suspicous despite the fact that the "Quantitation" view indicates now amplification.

Bacteria - C. pugettii culture CONTINUED (from 20090419)

Transferred 1mL of the culture to a 50mL conical containing 4mL of 1x Marine Broth and a couple crystals of biphenyl. Kept the cap loosened and incubated at 20C with shaking at 200RPM. Kept the existing culture in the incubator as well. No apparent growth.


20090420

gDNA Removal - Abalone RNA from 20090402 and 20090331

Transferred 50uL of RNA to fresh tubes and processed them using the Ambion Turbo DNA-free kit according to the manufacture's protocol.



20090419

Bacteria - C. pugettii culture


Rehydrated ATCC isolate according to directions. Added 5mL of 1x Marine Broth to a glass culture tube. Used 1mL of this to rehydrate ATCC sample and then added back into the culture tube. Added a few crystals of biphenyl, which had been exposed to UV for ~5mins prior. Incubated at 20C, no shaking. This was done at ~11:30AM.


20090417

Sequencing - Dungan isolates


Submitted two plates for sequencing. Each sample two times from each direction.

Plate #1

Plate #2




20090416

PCR - Two new Dungan isolates

Repeat of yesterday's PCR, but with AmpliTaq, less gDNA and 50uL rxn volume. PCR set up is here.


external image 20090416-02.JPG
Gel loaded and run by Steven. Not entirely sure of the loading order. However, it doesn't really matter...



Results: Still absolutely nothing.

UPDATE: Noticed on 20090713 that the reactions didn't have dNTPs. Probably explains why it didn't work!


20090415

PCR - Two new Dungan isolates from earlier today

Set up PCRs on:

MIE-14y
VNTc-1.2-C1/G10

Used Euk A/B and LABY A/Y primers. Anneal temp 50C. PCR set up is here.

NOTE: Due to the extremely low concentrations of gDNA from these two samples, I used a large amount of gDNA in the rxns. Check the PCR set up link for actual numbers.

external image 20090415.JPG
Lane 1 - Hyperladder (5uL)
Lane 2 - VNTc-1.2-C1/G10 (Euk primers)
Lane 3 - MIE-14y (Euk primers)
Lane 4 - H2O (Euk primers)
Lane 5 - H2O (Euk primers)
Lane 6 - VNTc-1.2-C1/G10 (Laby primers)
Lane 7 - MIE-14y (Laby primers)
Lane 8 - H2O (Laby primers)
Lane 9 - H2O (Laby primers)
lane 10 - 100bp Ladder

Results: Nada. Probably because of low [gDNA], but could also be due to PCR inhibitors in the gDNA. Will retry using Amplitaq and less gDNA.


qPCR - Rab7_SYBR primers on abalone RNA and DNased RNA

Attempt to find out if gDNA contamination exists iafter Ambion treatment. Previous test (on 20090414) suggests the QT Kit did not eliminate gDNA. PCR set up and plate layout here. Used Immomix and SYTO 13.

gDNA Isolation - Two new Dungan isolates

Isolated gDNA from the following two samples:
MIE-14y
VNTc-1.2-C1/G10

Samples were spun @ 16,000g @ RT for 2mins. No visible pellets in either sample. EtOH was removed. "Pellets" were washed in 1x PBS (pH=7.6) two times and then the Qiagen DNEasy Kit protocol was followed. Samples were incubated @ 55C with Proteinase K for ~2hrs.

external image 20090415%20gDNA%20SJW.jpg

Results: Both samples show really, really low quantities of gDNA.


20090414

PCR - Test QT Kit with No RT Abalone rxns from 20090408


Anneal 55C. PCR set up is here (bottom half of sheet).


external image 20090415.JPG

Lane 1 - Hyperladder
Lane 2 - gDNA
Lane 3 - cDNA pool
Lane 4 - No RT 06:5-31
Lane 5 - No RT 06:6-43
Lane 6 - No RT 08:3-5
Lane 7 - No RT 08:3-6
Lane 8 - No RT 08:3-7
Lane 9 - H2O
Lane 10 - H2O
Lane 11 - 100bp ladder

Results: No signal in the gDNA. Appropriate sized band in cDNA pool. Nothing in the water samples. HOWEVER, got bands in tow of the "No RT" rxns (08:3-6/7)!! It's odd that the gDNA didn't produce any signal, but there shouldn't be any signal in the No RT rxns. This indicates gDNA contamination. Should have also tested RNA and DNased RNA. Will test these in order to determine if that system is better at eliminating gDNA carryover.

PCR - Bay/Sea scallop gDNAs


Used higher annealing temps to improve primer specificity, compared to yesterday's results. PCR set up and plate layout is here.

external image 20090414-01.JPG

See the PCR/plate set up link for samples. Hyperladder is placed between every 12 samples.

Results: See this Google Spreadsheet for a summary of the 4 gels from the last two days.



20090413

PCR - Bay/Sea scallop gDNA isolated earlier today

Used 3 sets of reverse primers:
Bay_Actin_Rv0
Bay_Actin_Rv2
Sea_Actin_Rv2

Primers were slected based on information from Steven's notebook (#8, 12/30/2007-1/3/2008). Anneal temp 53C.
PCR set up here . Plate layout here .

Samples were run out by Steven the following day.
Gel 1 of 3
external image 20090413-02.JPG


Gel 2 of 3
external image 20090413-03.JPG

Gel 3 of 3
external image 20090413-04.JPG


Results:



gDNA Isolation - Bay/Sea Scallop and hybrid samples


gDNA was isolated using 500uL of 10% Chelex. Samples were heated @ 95C for 1hr w/periodic vortexing. Samples were then spun 16,000g for 5mins @ 4C. Stored @ -20C in Bay x Sea Scallop Project Box.


20090410

PCR - Abalone gDNA/RNA/cDNA w/new TOLLIP primer

Performed a new PCR on the three types of samples listed above with new TOLLIP primers. The new TOLLIP primers (H.discus_806_F/Ab_866_Rv) surround a putative intron. Thus, they will be useful for determining the presence of gDNA contamination in RNA/cDNA. Anneal temp 55C. PCR set up is here .

external image 20090413-01.JPG

Lane 1 - Hyperladder
Lane 2 - gDNA
Lane 3 - cDNA (QT)
Lane 4 - RNA (untreated)
Lane 5 - DNased RNA
Lane 6 - QT Kit, No RT
Lane 7 - H2O
Lane 8 - H2O

Results: This could possibly be the most confusing gel I've ever had the "pleasure" of running/analyzing, despite that it only has 7 samples.

No band in the gDNA sample, which could be explained by the intron size being too large for amplification with a basic polymerase. The cDNA worked as expected and contains a band of ~150bp. The RNA sample has a faint band of ~750bp. The DNased RNA sample has a band of ~400bp. The differences seen betweeen the gDNA (Lane 2), RNA (Lane 4) and DNased RNA (Lane 5) are truly bizarre. The No RT sample has no band. And, to top things off, one of the H2O samples is blank , but the other one has a band of ~1500bp! Ugh. How is all of this even possible?

PCR - Old Dungan isolates #1-35 w/EukA/B primers

Steven had me re-PCR the old Dungan isolates with the new EukA/B primers. Anneal temp 50C. PCR set up here (bottom half of sheet) .


NOTE: Sample #30 was not in the rack of tubes that Steven gave to me.

external image 20090410-01.jpg

Results:



PCR - Bay/Sea scallop hybrids


Because Rony's results from her PCR did not match her previous results for the positive controls, I ran the PCRs myself on the controls and the hybrids. Anneal temp 50C. PCR set up, samples, etc. are here.

external image 20090410.JPG


Results: The positive controls still do not match the results Rony got in two consecutive PCR attempts. However, the Bay_Actin_Rv2 and Sea_Actin_Rv3 primers do result in clearly distinguishable differences between bay and sea scallop gDNA. Lane 2 (bay gDNA/bay actin primer) has a large, ~1000bp band and lane 7 (sea gDNA/sea actin primer) produces a ~300bp band. Unfortunately, this does provide us with any useful info. Something needs to be reworked (i.e. possibly new target gene) in order to start getting some useful results.

However, the results we did get definitely confirm that the hybrids are NEITHER hybrids nor bay scallop, due to the fact that no bands are present in any other sample than the bay scallop gDNA samples.


20090409

PCR - Dungan isolates from 20090402 with Euk primers

Did PCR with new Euk primers designed by Steven. Should be one step higher taxonomically. PCR set up is here. Aneal temp 50C.

external image 20090409.JPG

Lane 1 - Hyperladder
Lane 2 - 19t
Lane 4 - 13t
Lane 6 - 17t
Lane 7 - 100bp ladder
Lane 8 - 1.5t
Lane 10 - 1.2t
Lane 11 - Hyperladder
Lane 12 - 11t
Lane 14 - H5
Lane 15 - 100bp ladder
Lane 16 - 12t
Lane 18 - H2O
Lane 19 - H2O
Laen 20 - Hyperladder

Results: The new EukA/B primers worked wonderfully. The brightest band in each lane was excised and purified using Millipore DA spin columns. These will be stored and sequenced at a later date.

qPCR - Check DNased abalone RNA (by Lisa) for gDNA

qPCR was performed with 16s_sybr primers on the DNased RNA that Lisa did. Annel temp 55C. Sample set up and plate layout is here.

Results: Still got signals in all of the samples, including the waters. Personally, I think the primers are contaminated or are forming crazy dimers. Lisa came by and picked up cDNA to run other genes on.

qPCR - Repeat (modified) of yesterday's abalone cDNA check

qPCR was performed with 16s_sybr primers on a subset of the "No RT" cDNA rxns from yesterday at both 55C and 60C. Sample set up and plate layout is here.

Results: Still getting signals in the "No RT" rxns and possibly in the waters. Cut run short to start another. Will test Lisa's previously DNased RNA.


20090408

qPCR - Abalone cDNA (QT) from earlier today

qPCR was performed with 16s_sybr primers on a subset of the cDNA rxns and all of the "No RT" rxns from earlier to detect the presence of contaminating gDNA. qPCR was also performed with the Rab7_sybr primers on a subset of the cDNA rxns to check that the assay would work. The qPCR set up sheet/plate layout is here. Annealing temp = 55C.

Results: Apparently the gDNA wipeout step did NOT work! Also, some signal in the water samples.

cDNA - Abalone RNA from 20090331 & 20090402


cDNA was made from the above RNA samples using the Qiagen Quantitect RT Kit. The samples were laid out in a PCR plate. 274.2ng of RNA was used in the rxn for each sample, based on the lowest concentration RNA sample (08:3-20) to equalize all the samples. The Genomic Wipeout step of the kit requires 2uL of Genomic Wipeout enzyme/buffer to be added to 12uL of an RNA sample, so the calculations were done and can be found here. A check mark on the calculation sheet indicates that the water and then the RNA was added to the appropriate wells. Those with two check marks were used for a "No RT" rxn and thus, have duplicate wells (see plate layout).

The plate was mixed, spot spun, uncubated at 42C for 2mins and immediately placed on ice.

The RT and No RT master mixes were set up on ice and then added to the respective wells (see sheet here).

UPDATE: cDNA plate was discarded 20120320 by SJW.


20090406

PCR - New Dungan isolates


Repeat of PCR from 20090403, but using AmpliTaq and 50C annealing temp. PCR set up is here.

external image 20090407.JPG

Lane 1 - Hyperladder
Lane 2 - 19t
Lane 3 - 100bp ladder
Lane 4 - 13t
Lane 6 - 17t
Lane 8 - 1.5t
Lane 9 - Hyperladder
Lane 10 - 1.2t
Lane 12 - 11t
Lane 13 - 100bp ladder
Lane 14 - H5
Lane 16 - 12t
Lane 17 - 100bp Ladder
Lane 18 - H2O
Lane 19 - H2O
Lane 20 - Hyperladder

Results: Nothing amplified! Possibly due to age of polymerase (?); over a year old. Will wait to repeat for new primers to arrive (EukA/B).


20090403

PCR - New Dungan isolates

Ran PCR with GoTaq on the new Dungan isolate gDNA from yesterday. PCR set up is here. 53C annealing temp.

external image 20090403.JPG

Lane 1 - Hyperladder
Lane 2 - 17t
Lane 4 - H5
Lane 6 - 12t
Lane 7 - Hyperladder
Lane 8 - 1.5t
Lane 10 - 1.2t
Lane 12 - 11t
lane 13 - Hyperladder
Lane 14 - 13t
Lane 16 - 19t
Lane 18 - H2O
Lane 19 - Hyperladder
Lane 20 - H2O

Results: All the bands present are a bit larger than 400bp. However, the bottom band in the H5 sample is largerer than any band present in any other samples. Additionally, the largest band in the H5 sample is between 800-1000bp. Bands were cut out from H5 (two bands: Top, Bottom), 1.5t, 1.2t, 13t. These will be purified and sequenced.

It's also interesting to note that the bands present in this gel are found in the same samples as the last time this analysis was done. See Kevin's Notebook, 20090212.

qPCR - Abalone RNA, check for gDNA

Abalone RNA isolated from this week was qPCR's with 16s primers to determine if gDNA contamination was present. PCR/plate layout is here.

Results: All samples appear to have gDNA contamination. Will use Qiagen QT Kit for cDNA.




20090402

gDNA Isolation - New Dungan isolates

gDNA was isolated from the following using the Qiagen DNEasy Kit:

xCvC-19t (3/26/2009)
xCvE-13t (3/16/2009)
xCvC-17t (3/18/2009)
UNTc-1.5t (3/18/2009)
VATm-1.2t (3/16/2009)
xCvC-11t (3/18/2009)
BC05Ca18c/H5 (3/27/2009)
xCvC-12t (3/16/2009)

500uL was of each sample was transferred to a 1.5mL snap cap tube. The samples were pelleted by spinning 5 mins @ 16,000g and washed 2x w/ 1x PBS pH=7.6. Samples were then processed according to Qiagen protocol. Initial digestion with Proteinase K was 2hrs.

external image 20090403%20gDNA%20SJW.jpg


RNA Isolation - Abalone digestive gland samples

Total RNA was isolated from the following abalone digestive gland samples using the RNA Powersoil Kit, according to their protocol:

08:3-17
08:3-18
08:4-2
08:4-3
08:4-4
08:4-5
08:4-7
08:4-8
08:4-11

Notes: 08:4-5 took hours to wash the column. 08:4-4 ended up with a dark RNA pellet and the reuspended RNA is discolored (brown-ish).

external image 20090402%20RNA%20SJW.jpg

external image 20090403%20RNA%20SJW.jpg
Results: All the RNA looks good except for 08:4-4, but that isn't surprising (see note above). Samples were stored @ -80C in the boxes from which the tissue samples came.



20090331

RNA Isolation - Abalone digestive gland samples

Total RNA was isolated from the following abalone digestive gland samples using the RNA Powersoil Kit, according to their protocol:

08:3-6
08:3-7
08:3-8
08:3-9
08:3-14
08:3-15
08:3-16
08:3-24

external image 20090331%20RNA%20SJW.jpg

Results: RNA looks great. Stored @ -80C in box where samples came from.


20090327

RNA Isolation - Abalone digestive gland samples


Total RNA was isolated from the following abalone digestive gland samples using the RNA Powersoil Kit, according to their protocol:

08:3-11
08:3-12
08:3-13
08:3-19
08:3-20
08:3-21
08:3-22
08:3-23

Notes: After phase separation, 3-11 and 3-12 had milky/cloudy aqueous phases. These two samples were subjected to another 10min spin @ 2500g, but this spin made no difference in their appearance. Other samples were clear or slightly translucent at worst.


external image 20090330%20RNA%20SJW.jpg

Results: RNA looks great in nearly all of the samples. RNA has been stored @ -80C in the same box from where the tissue was taken.



20090324

qPCR - Virginica cDNA (see workup sheet below for more info regarding cDNA)

Set up qPCR using the following Virginica primers provided by Mac:

Cv_BgBL
Cv_BGBP
Cv_CysB
Cv_HMG
Cv_HSP70

Here is the qPCR workup sheet .



20090316

mRNA Sample Submission Hard clam gill #1 mRNA from 20090313

Submitted four samples (4uL of each) for Agilent Bioanlyzer:

SR01 = total RNA
SR02 = Ambion kit x 1
SR03 = Ambion kit x 2
SR04 = PolyA Tract kit

Results:
external image 20090319_Hclam_gel_LABELED.jpg


20090313

mRNA Isolation - hard clam gill #1 continued from yesterday


Precipitation was continued from yesterday. Samples were resuspended in 25uL of The RNA Storage Solution (from PolyAPursit Kit) and spec'd. Samples were stored @ -80C in Sam's RNA box.

external image 20090313%20RNA%20SJW.jpg


20090312

mRNA Isolation - hard clam gill #1 DNased RNA from today

DNased RNA from earlier today was split into four equal parts (175uL = 39.8ug). Three will be used for mRNA isolation and the fourth will remain as total RNA. Three of these were precipitated according to Ambion PolyAPurist Protocol: 1/10 volume 5M ammonium acetate, 1uL glycogen and 2.5 volumes of 100% EtOH. Incubated @ -80C for 30 mins. One sample was processed with the Promega PolyA Tract kit. The remaining two samples were processed according to PolyAPurist Protocol. Of those two, one of the samples was processed a second time to evaluate the effectiveness of running a sample through the PolyAPurist Protocol twice.

mRNA samples were precipitated O/N @ -20C according to the PolyAPurist Protocol.

RNA Precipitation - Hard clam gill #1 RNA from 20080819

DNAsed RNA using Ambion Turbo DNA-free kit, following the rigorous procedure. Diluted total RNA to 0.2ug/uL (Vf = 720uL). Added 1uL DNase and incubated the tube @ 37C for 30mins. Added an additional 1uL DNase and continued incubated for 30mins. Added 0.2 volumes of DNase Inactivation Reagent (158.4uL) and incubated at RT for 10mins with periodic mixing. Pelleted inactivation reagent according to protocol and transferred supe (DNA-free RNA) to clean tube.

external image 20090312%20RNA%20SJW.jpg

Results: RNA looks really nice. Have a large quantity of RNA (700uL x 0.2275ug/uL = 159.25ug). Will split into four equal parts and isolate mRNA from 3 of the 4. Those three will be:

1. Ambion kit x 1
2. Ambion kit x 2
3. Promega PolyA Tract kit.



20090309

qPCR - New Vtub_IGS primers : V.tub Control vs. Autoclaved gigas samples (see 20090224)




20090302

qPCR - Repeat of qPCR from earlier today with fresh primer working stocks

This is an exact repeat of the qPCR from earlier today, but using a fresh working stock of the Vtub_16s_V3 primers. The plate layout/qPCR workup is here.

Results: Same as earlier today. Must be a bacterial contaminant somehwere that these 16s primers are picking up. Will order IGS primers that are species specific found in Lee et al. 2002.

qPCR - Repeat of 20090227 qPCR with clean water

This is an exact repeat of the qPCR from Friday, but using a fresh aliquot of water for preparation. The plate layout/qPCR workup is here.

Results: Same as Friday. Fluorescence comes up way too fast and there is contamination present in in the water. Will repeat with a fresh preparation of the primer working stocks.




20090227

qPCR - New 16s primers for V.tubiashii Control vs. Autoclaved gigas samples (see 20090224)

qPCR was performed using SensiMix/SYBR "kit" with DNAsed RNA samples from 20090224. This qPCR used the new V.tub_16s_V3 primers in hopes of getting better amplification; both in signal intensity and elimination of the double peak seen in the melting curves from 20090224. The plate layou/qPCR workup is here.

Results: Fluorescence comes up WAY too early; at like the 5th cycle! Also, there are two peaks in the melting curves. Additionally, there is a signal in the two water samples and the melting curve for this contamination matches up with one of the melting cure peaks seen in the actual sample melting curves. So, there is some sort of contamination somehwere. Will repeat this using a clean water for the master mix and hope the problem goes away.


20090225

qPCR - Replicate of V.tubiashii Control vs. Autoclaved gigas samples (see yesterday)

This is a repeat of the qPCR from yesterday, but without the 16s and OmpW primer sets due to double peaks in melting curves yesterday. Plate layout/qPCR workup is here.

Results: Similar to yesterday's results, the amplification looks a bit odd when viewing on a log scale. However, the linear scale curves look to be normal. Melting curves look good for all genes examined and there is not any detectable gDNA in the RNA samples. Excellent...


20090224

qPCR - V.tubiashii Control vs. Autoclaved gigas samples (see below)

qPCR was performed using SensiMix/SYBR "kit" with DNAsed RNA samples and cDNA made earlier today. The plate layout/qPCR workup is here.

Results: Generally, the amplification looks a bit odd when viewing on a log scale. However, the linear scale curves look to be normal. There doesn't appear to be any gDNA contaminiation in the RNA samples, BUT the Vtub_16sV2 primers used on the RNA samples also do not produce much of a signal in the cDNA either. The melting curves for the 16s and the Vtub_OmpW primer sets have multiple peaks. Will likely order new 16s primes, due to weak signal.

Will redo qPCR on the DNAsed RNA to make sure that the lack of detectable signal is due to lack of gDNA and NOT because the 16s primers don't work. Also will repeat in order to have a replicate of the other samples.

Reverse Transcription - V.tubiashii DNAsed RNA (from yesterday)


Set up the MMLV RT rxns with random primers using ~833ng DNAsed RNA (prepared yesterday) according to the Promega MMLV Product Insert. This procedure is slightly different than what is in our lab protocol for RT rxns. Here is the workup for the rxns. cDNA was stored @ -20C in the "Vibrio" box.

20090223

RNA Isolation - V.tubiashii samples from autoclaved gigas exposure (from 20081218)

RNA was isolated from the Control and V.tub+gigas samples from the 0, 1, & 24hr time points using 1mL TriReagent. No visible pellets. Used 20uL of 0.1%DEPC-H2O to resuspend RNA. Incubated @ 55C, 5mins. Spec'd.

external image 20090223%20RNA%20SJW.jpg

Results: RNA looks OK, but not great. For the "V.tub + gigas t=1" sample, the third spec reading is correct. The first two had the air bubble error.

DNAse Treatment - V.tubiashii total RNA (see above)

1ug of RNA in a volume of 12uL was DNAsed using the Ambion DNA-free Kit according to their protocol. RNA was transferred to a fresh tube and stored @ -80C in Sam's RNA Box #1.

20090220

Epigenetics Experiment - Gigas treatment

Set up two containers with 3L seawater and 4 gigas in each container. The treated sample contained:
5mL of 5N NaOH
5mL of Bl
10mL of 1x LB
10mL of 95% EtOH
1mL of form

Both containers were covered, stored in the fume hood and incubated over the weekend. Experiment was started at 3:15PM.

Epigenetics Experiment - Vibrio culture CONTINUED (from yesterday)


Culture did not grow. Colonies probably too old. Plate also failed to grow any colonies. Will streak out fresh cultures from frozen stock.

20090219

Epigenetics Experiment - Vibrio culture

Inoculated 500mL of 1x LB + 0.1% NaCl with V. tubiashii. Incubated O/N, 25C, 200RPM. Also, streaked a fresh LB+0.1% NaCl plate with V.tubiashii from same plate used to inoculate liquid culture. Incubated plate O/N @ 37C.


20090218

Mass Spec

Vibrio samples (2-1 and 2-2) from 20090212 were submitted to the mass spec facility.


20090212

Trypsin digestion - Vibrio 2D spots CONTINUED (from yesterday)


Sample prep was continued from yesterday. SpeedVac'd final, pooled elutions for 1.5hrs. Stored tubes @ -80C. Will submit samples 2-1 and 2-2 for mass spec analysis.


20090211

Trypsin digestion - Vibrio 2D spots from 20081217


Samples were prepared according to Goodlett Lab protocol and incubated O/N on LabQuake.



20090206

mRNA - Submission for Agilent Bioanalyzer


Submitted 4uL (108ng/uL) of mRNA from gigas gill (from DATE!; sometime in late August) to the Center for Array Technologies (CART) at UW. Sample was labeled "SR01".

Results:

external image 20090209_Gigas_LABELED.jpg

Results were received on 20090210. Comment from CAT staff member was that it looked "pretty good." Here are links to the gel (look for SR01), the electoropherogram of SR01, and the EGRAM of the ladder.

20090123

mRNA - Precipitation continued from yesterday

Samples were pelleted and washed with 70% EtOH according to Ambion PolyA Purist protocol. Pellets were resuspended in 10uL of The RNA Storage Solution (included in the Ambion PolyA Purist Kit).

external image 20090123%20RNA%20SJW.png

Results: The gill mRNA looks great! Good yield and good ratios. Hemocyte mRNA looks kinda rough and a very low yield (which was to be expected).


20090122

mRNA Isolation - Hard Clam gill and hemo RNA

mRNA was isolated according to Ambion PolyA Purist protocol. After mixing samples with resin, samples were incubated @ RT for 1hr. Samples were washed per the protocol. However, the hemo sample was not clearing from the spin columns with the protocol-directed 3 min. spins. The column had to be spun up to 15 mins. in order for the column to clear. :(

external image 20090122%20RNA%20SJW-02.png

Results: The gill mRNA looks good (~1.8ug). The concentration of the hemo sample is extremely low and is below the error threshold for the NanoDrop, but it may not be that bad. The samples are in a relatively large volume (~200uL) and the yield is expected to be very small. So, I will precipitate these samples O/N @ -20C according to Ambion PolyA Purist protocol in order to concentrate them.

RNA - Hard clam hemo RNA (from 20090121)

The two RNA samples from yesterday were precipitated and washed according to the Ambion PolyA Purist protocol and resuspended in 50uL of 0.1% DEPC-H2O.

external image 20090122%20RNA%20SJW.png

Results: RNA readings look better than they did prior to precipitation. The hemo RNA samples will be combined with previous hemo RNA samples and mRNA will be isolated using the Ambion PolyA Purist Kit.


20090121

RNA Isolation - Hard clam hemo (from 20090121)

The 8 hemo samples were pooled and the 4 gonad/d.g. samples were pooled. RNA was isolated. The homo sample was resuspended in 100uL of 0.1%DEPC-H2O and the gonad/d.g. sample was resuspended in 50uL 0.1%DEPC-H2O. RNA was precipitated O/N @ -20C according to Ambion PolyA Purist protocol in preparation for mRNA isolation.

external image 20090121%20RNA%20SJW.png


20090120

Bleeding/Tissue Collection - Hard Clams

Hemos were collected from the remaining 12 clams as before and stored @ -80C. NOTE: 4 hemocyte samples were extremely cloudy. Possibly not hemos? Maybe gonad/d.g.? Gill tissue was collected from 5 clams and stored @ -80C.

RNA - Precipitation of Hard Clam Hemo RNA from 20090116

RNA isolated on 20090116 was precipitated over the weekend @ -20C. Samples were treated according to Ambion PolyA Purist protocol and resuspended in 100uL of 0.1% DEPC-H2O. Samples were stored in the red "hard clam" box @ -80C.

external image 20090120%20RNA%20SJW.png
Results: 260/280 look good for both gill and hemo samples. 260/230 looks OK for gill, but looks horrible (as usual) for hemos.


20090116

RNA Isolation - Hard clam gill, hemos

RNA was isolated from hard clam gill (0.08g; from 20090109) and from hard clam hemos (from 20090114). One "unknown" sample (from 20090114) was also processed. Pellets were resuspended in 50uL of 0.1%DEPC-H2O. Hemo samples were pooled. Samples were spec'd.

external image 20090116%20RNA%20SJW.PNG

Results: 260/280 looks OK for hemos. The gill sample has excellent 260/280 and pretty good 260/230. Samples will be reprecipitated over the weekend @ -20C according to Ambion PolyA Purist protocol in preparation for mRNA isolation.


20090114

Bleeding - Hard Clams

Bled 7 clams from 20090108 and 20090109. Bled clams using a 23g 1.5 needle on a 3mL syringe. Fluid was gathered and ranged from ~0.4-1.0mL. Hemolymph was transferred to individual 1.5mL snap cap tubes and spun @ 100g for 30mins @ 4C. Most of the supe was removed, but left ~100uL in each tube to avoid disturbing any pellet. Samples were stored @ -80C in the red box with previous hard clam hemo samples.
NOTE: One sample was EXTREMELY cloudy. Likely not hemos.

RNA - Precipitation continued from yesterday

Transferred supe to a fresh tube and added 1mL 70% EtOH to remaining pellet. Spun samples max speed @ 4C 30 mins. Removed supe and washed pellets with 1mL 70% EtOH. Spun max speed 10 mins. Removed supe . Resuspended the "supe" sample in 50uL 0.1%DEPC-H2O and the "pellet" sample in 100uL 0.1%DEPC-H2O.

external image 20090114%20RNA%20SJW.png

Results: 260/280 ratios look good. The 260/230 ratios are still horrible. Total yield from these two samples are ~5ug. Will get more hemolymph from clams in order to use more total RNA in the mRNA isolation to maximize cost saving.


20090113

RNA - Reprecipitation of hard clam RNA from yesterday

Because of the relatively large size of the pellets vs. the amount of RNA, I think another round of precipitation would be best to help remove additional residual salt carryover. Will precipitate O/N according to Ambion PolyA Purist protocol. RNA pellets were resuspended in 250uL of 0.1%DEPC-H2O and precipitated O/N @ -20C.

NOTE: Upon adding 100% EtOH to sample, the solution turned very cloudy and a white precipitate immediately formed inside the tube. I do not think this precipitate is RNA. Tomorrow, before spinning the tube, I will transfer the supe to a fresh tube and process both tubes simultaneously. Hopefully this will remove/eliminate most of the excess salt or whatever seems to be forming the pellet.





20090112


Bleeding - Hard Clams

Bled 8 clams from 20090108 and 20090109, #4, 6, 8, 15, 16, 17, 21, 26. Bled clams using a 23g 1.5 needle on a 3mL syringe. Fluid was gathered and ranged from ~0.4-1.0mL. Hemolymph was transferred to individual 1.5mL snap cap tubes and spun @ 100g for 30mins @ 4C. Most of the supe was removed, but left ~100uL in each tube to avoid disturbing any pellet. Samples were stored @ -80C in the red box with previous hard clam hemo samples.

Pellets were apparent in all 8 samples, whereas they had not been noticeable before in last week's bleeds.

Also, 3 clams were found with cracked shells, but alive, including the one pictured below that is split open entirely.

external image 20090112%20M.mercenaria%20cracked-01.JPG

external image 20090112%20M.mercenaria%20cracked-02.JPG


RNA Isolation - Hard Clam hemolymph from 20090108, 20090109


1mL of TriReagent was used to isolate RNA from 3 combined tubes of hemolymph. This resulted in 10 total RNA preps. Pellets were resupsended in 100uL of 0.1% DEPC-H2O and pooled into a single tube and NanoDropped.

external image 20090112%20RNA%20SJW.jpg

Results: RNA solution looked very cloudy and contains a fair amount of insoluble "stuff". 260/280 ratios also looked bad. Will precipitate O/N according to Ambion PolyA Purist Kit before isolating mRNA tomorrow.



20090109

Bleeding - Hard Clams

Bled 24 hard clams using a 23g 1.5 needle on a 3mL syringe. Fluid was gathered and ranged from ~0.4-1.0mL. Hemolymph was transferred to individual 1.5mL snap cap tubes and spun @ 100g for 30mins @ 4C. Most of the supe was removed, but left ~100uL in each tube to avoid disturbing any pellet. Samples were stored @ -80C in the red box wiht previous hard clam hemo samples. Clams were numbered and transferred to a holding tank. Gill and mantle tissue was collected from 10 of the clams. The collected tissue and the rest of the carcasses were stored @ -80C.


20090108

Bleeding - Hard Clams

Bled 6 hard clams using a 23g 1.5 needle on a 3mL syringe. Fluid was gathered and ranged from ~0.4-1.0mL. Hemolymph was transferred to individual 1.5mL snap cap tubes and spun @ 100g for 30mins @ 4C. Most of the supe was removed, but left ~100uL in each tube to avoid disturbing any pellet. Samples were stored @ -80C in the red box with previous hard clam hemo samples. Clams were numbered and transferred to a holding tank.


20090107

PCR - Dungan Isolates

Samples (in Chelex) were vortexed and heated @ 95C for 30mins with periodic vortexing. Tubes were spun max speed @ 4C for 2 mins to pellet Chelex. Set up PCR using Immomix master mix. Anealing temp. = 56C. PCR set up here.


external image 20090108.png


Lane 1 - 100bp ladder
Lane 2 - xCvC-11t
Lane 3 - xCvC-12t
Lane 4 - xCvC-17t
Lane 5 - VNTc-12-C1/G10
Lane 6 - BC05Ca-18t/H5
Lane 7 - VATm-1.2t
Lane 8 - VNTc-1.5t
Lane 9 - Neg. Control

Results: PCR seems to have worked for some of the samples. The bottom-most band in lanes 4, 5, 6, 9, & 10 were cut out and stored in "Sam's Misc. -20C Box". Date is 1/8/2009, since this PCR ran O/N.



20090106

PCR - Dungan Isolates

All samples , except xCVC-11t, are already in Chelex. For xCvC-11t, pipetted a shunk of cells/tissue from source tube. Volume of liquid (EtOH) was ~75uL. Added this to screw cap tube containing 300uL of 10% Chelex solution. Vortexed and incubated @ 95C for 30mins. Vortexed and incubated other samples at 95C for 5mins. Set up PCR with AmpliTaq. Anneal temp. = 53C. PCR set up here.


external image 20090106.png

Lane 1 - 100bp ladder
Lane 2 - xCvC-11t
Lane 3 - xCvC-12t
Lane 4 - xCvC-17t
Lane 5 - VNTc-12-C1/G10
Lane 6 - BC05Ca-18t/H5
Lane 7 - VATm-1.2t
Lane 8 - VNTc-1.5t
Lane 9 - Neg. Control


Results: Ladder is degraded and there are no bands in any lane. Will repeat and try to duplicate Steven's results from 20080916.


20090102

SDS/PAGE, Western Blot - Test of HSP70 Ab on heat stressed shellfish for FISH441


Pacific oysters, a mussel, barnacles and a clam (sp. ?) were transferred from the holding tank to a large beaker with sea water which was placed into a 37C water bath. The shellfish were incubated in this water bath for ~3hrs. Tissues were collected from each, transferred to a 50mL conical tube and immediately placed in a dry ice/ethanol bath:

Oyster - gills, muscle and mantle
Clam - whole clam
Mussel - whole mussel
Barnacles - whole barnacles

0.02 - 0.07g of each tissue were weighed, added to a 1.5mL tube containing 0.5mL CelLytic MT + protease inhibitors and homogenized. For the barnacles, ~8 barnacles were transferred to a weigh boat and smashed with a hammer. This was then transferred to a 1.5mL tube with CelLytic MT + protease inhibitors. Tubes were spun @ max speed @ 4C for 10mins. Supe was transferred to a fresh 1.5mL snap cap tube. 15uL of each sample was transferred to a screw cap tube containing 15uL of 2X reducing sample buffer. Tubes were boiled for 5mins and then spun for 1min @ max speed @ 4C. Samples were loaded on Pierce 4-20% Tris-HEPES SDS PAGE gels and run 150V for 45mins. 10uL of SeeBlue Plus ladder was also loaded on the gel.

Gel, membrane, and blotting paper were soaked for 15mins in transfer buffer (Pierce recipe w/methanol). Proteins were transferred to nitrocellulose membranse for 30mins @ 20V. Gel was stained for ~40mins in Coomassie stain and then destained w/10% acetic acid solution until bands were clearly visible.

external image 20090102%20PAGE.JPG

Western blotting was done according to Invitrogen WesternBreeze Chromogenic (anti-mouse) protocol. Primary HSP70 Ab was used at a 1:3000 dilution (per the Meistertzheim et al. paper).

external image 20090102%20labeled.png