20091231
qPCR - BB & DH cDNA (from 20091223)
qPCR was set up on these cDNAs using the following primers:
FP008495.p.cg.6 ("GSTO1", "Glutathione S-transferase omega-1") - This was upregulated in BB SOLiD data.
FP008495.p.cg.6 ("GSTO1", "Glutathione S-transferase omega-1") - This was upregulated in BB SOLiD data.
These were run in duplicate to take up a full PCR plate.
qPCR set up and plate layout can be found here.
Results:
20091230
qPCR - BB & DH cDNA (from 20091223)
qPCR was set up on these cDNAs using the following primers:
CU684779.p.cg.6 ("SEMSA", "Semaphorin-SA") - This was upregulated in BB SOLiD data.
FP004879.p.cg.6 ("TIMP3", "Metalloproteinase inhibitor 3") - This was upregulated in BB SOLiD data.
qPCR set up and plate layout can be found here.
Results:
qPCR - BB & DH cDNA (from 20091223)
qPCR was set up on these cDNAs using the following primers:
AJ582629.p.cg.6 ("DEF1", "Defensin 1") - This was upregulated in BB SOLiD data.
CB617519.p.cg.6 ("RETST", "All-trans retinol") - This was upregulated in BB SOLiD data.
qPCR set up and plate layout can be found here.
Results:
20091229
qPCR - BB & DH cDNA (from 20091223)
qPCR was set up on these cDNAs using the following primers:
FP010108.p.cg.6 ("DJB12", "DnaJ homolog subfamily B member 12") - This was upregulated in DH SOLiD data.
AJ565670.p.cg.6 ("TOP1", "DNA topoisomerase 1") - This was upregulated in BB SOLiD data.
qPCR set up and plate layout can be found here.
Results:
Alaska sockeye salmon sampling (with Seebs): Family #13
Juvenile sockeye salmon were subjected to initial heat stress of 18C. 10 fish were weighed, measured and then snap frozen in LN2. Samples were transferred to a freezer box labeled "AL Sockey Family #13" and stored @ -80C.
Here is the spreadsheet with all the pertinent info.
qPCR - BB & DH cDNA (from 20091223) and Emma primer sets for testing
qPCR was set up on these cDNAs using the following primers:
EW778389.p.cg.6 ("DPGN", "Serine protease inhibitor dipetalogastin") - This was upregulated in DH SOLiD data.
FP001672.p.cg.6 ("PGSC1", "Peptidoglycan-recognition protein SC1a/b") - This was upregulated in DH SOLiD data.
Included three primer sets of Emma's (matrillin, beta tub and chaperonin). These were set up with no template and done in duplicate.
qPCR set up and plate layout can be found here.
Results: Emma's "beta tub" primers show some weird fluorescence, however none of the primer sets show any thing in the melting curve analysis.
20091228
qPCR - BB & DH cDNA (from 20091223)
qPCR was set up on these cDNAs using the following primers:
CU994646.p.cg.6 ("CATL", "Cathepsin L") - This was upregulated in DH SOLiD data.
ES789598.p.cg.6 ("GSTA", "Glutathione S-transferase A") - This was upregulated in DH SOLiD data.
qPCR set up and plate layout can be found here.
Results:
qPCR - BB & DH cDNA (from 20091223)
qPCR was set up on these cDNAs using the following primers:
CU988730.p.cg.6 ("TIMP3", "Metalloprotease inhibitor 3") - This was upregulated in DH SOLiD data.
CU990442.p.cg.6 ("CALL", "Calmodulin-like protein") - This was upregulated in DH SOLiD data.
qPCR set up and plate layout can be found here.
Results:
qPCR - BB & DH cDNA (from 20091223)
qPCR was set up on these cDNAs using the following primers:
AM861391.p.cg.6 ("BDEF", "Big Defensin") - This was upregulated in DH SOLiD data.
AM904566.p.cg.6 ("GNRR2", "Gonadotropin-releasing hormone II receptor") - This was upregulated in DH SOLiD data.
qPCR set up and plate layout can be found here.
Results:
20091224
qPCR - BB & DH cDNA (from yesterday)
qPCR was set up on these cDNAs using the following primers:
AM855874.p.cg.6 ("CP17A", "Steroid 17-alpha-hydroxylase/17") - This was upregulated in DH SOLiD data.
AM857078.p.cg.6 ("CIQT4", Complement C1q tumor necrosis factor-related protein 4") - This was upregulated in DH SOLiD data.
qPCR set up and plate layout can be found here.
Results:
qPCR - BB & DH cDNA (from yesterday). EW778094, AJ422120.p.cg.6 primers
qPCR was set up on these cDNAs using the following primers:
EW778094 ("Ficolin 3") -
AJ422120.p.cg.6 ("MDR49", "Multidrug resistance protein homolog 49") - This was upregulated in DH SOLiD data.
qPCR set up and plate layout can be found here.
Results:
Preliminary results show that the EW778094 primer set looks bad; bad fluorescence profile and poor melt curves. Primers may require optimization.
20091223
Sequencing - Mac methylation samples, Sam rhodopsion samples, Lisa samples
Samples were submitted for sequencing. Mac prepped all Roberts Lab samples excluding the Opsin VMC gel slice 2 from 20091217-02. The gel slice was purified with Millipore spin columns. The sample was diluted 1:1 with H2O and submitted for sequencing, one time from each direction using the Sep_Op_Fw2/Rv2 primers.
Plate layout can be found here on sheet labeled "20091223".
qPCR - BB & DH cDNA (from earlier today)
qPCR was set up on these cDNAs using HMGP_5'/3' and SPI_5'/3' primers.
qPCR set up and plate layout can be found here.
Results:
Reverse Transcription - BB & DH DNased RNA (from 20090514)
Made a fresh, double batch (50uL rxn instead of 25uL) of cDNA according to Promega MMLV protocol using oligo dT primers. cDNA was put into a plate for faster qPCR loading.
cDNA calcs and plate layout are here. Briefly, RNA and oligo dTs were combined, brought up to 37uL, heated @ 70C for 5mins and immediately placed on ice. RT master mix was made (
RT master mix calcs are here), 13uL was distributed to each well. Samples were incubated @ 42C for 1hr and then 95C for 5mins.
UPDATE: cDNA plate was discarded 20120320 by SJW.
20091217
PCR - Sepia cDNA
This is an exact repeat of the PCR from yesterday, due to inconsistencies between repeated PCRs from yesterday and earlier today.
Here's yesterday's workup
Results:
Gel Loading (from left to right):
Opsin Primers (lanes 2-10), Rhodopsin Primers (same loading order, lanes 12-20)
1 - 100bp ladder
2 - retina
3 - fin
4 - 4th arm
5 - dorsal mantle center
6 - dorsal mantle side
7 - ventral mantle center
8 - ventral mantle side
9 - H2O
10 - H2O
Opsin Primers
Single bands in retina, fin and ventral mantle center samples. Negative controls are clean. Bands will be excised and stored @-20C in Sam's "Purified Inserts" box for eventual sequencing. Tubes are labeled with a "2" to differentiate them from the gel run earlier today.
Rhodopsin Primers
Single bands in retina and fin. Double bands in ventral mantle center sample. Second band is very faint is ~700bp. Negative controls are clean. Bands, including the faint 700bp VMC, will be excised and stored @-20C in Sam's "Purified Inserts" box for eventual sequencing. Tubes are labeled with a "2" to differentiate them from the gel run earlier today.
20091216
PCR - Sepia cDNA
This is an exact repeat of the PCR from yesterday, due to inconsistencies between repeated PCRs from yesterday and earlier today.
Here's yesterday's workup. Samples were stored @ 4C O/N after completion of PCR. Gell was run on 20091217.
Results: This is getting embarrassing. Opsin results are same as yesterday (good!), but Rhodopsin results are slightly different than yesterday's AND the day before yesterday's results!
Gel Loading (from left to right):
Opsin Primers (lanes 2-10), Rhodopsin Primers (same loading order, lanes 12-20)
1 - 100bp ladder
2 - retina
3 - fin
4 - 4th arm
5 - dorsal mantle center
6 - dorsal mantle side
7 - ventral mantle center
8 - ventral mantle side
9 - H2O
10 - H2O
Opsin Primers
We see a band in the retina, fin, 4th arm, & ventral mantle center samples as we did yesterday. This replicates yesterday's PCR results, which is good. Negative controls are clean. Bands will be excised and stored @-20C in Sam's "Purified Inserts" box for eventual sequencing.
NOTE: This gel was run on 20091217 and the tubes are dated as such!
Rhodopsin Primers
A single band is present in the retina, dorsal mantle center and ventral mantle center samples. We see TWO bands in the fin sample. These results differ from yesterday's in that yesterday, a single band was present ONLY in the retina and fin samples. Two days ago there was a single band in each of the retina and fin samples and two bands in the ventral mantle center sample. Bands will be excised and stored @-20C in Sam's "Purified Inserts" box for eventual sequencing.
NOTE: This gel was run on 20091217 and the tubes are dated as such!
I am going to repeat these PCRs again. I can't stand the fact that I am getting such freaking inconsistent results in an extremely simple PCR. Aaargh!
PCR - Sepia cDNA and DNased RNA
Set up PCR on recent Sepia cDNA and DNased RNA samples using both S. officianalis_rhodopsin_F, R primers & Sep_op_F2, R2 primers. After yesterday's intirguing PCR results, we need to confirm that the DNased RNA is free of contaminating gDNA. Additionally, we want to excise bands from the cDNA samples for sequencing.
PCR set up is here. RNA was prepped as though making cDNA; diluted 0.2ug of DNased RNA in a final volume of 25uL. Used 1uL of this for PCRs.
Rhodopsin primers require 3mM Mg2+ in the PCR rxn and the opsin primers require 2mM Mg2+ in the PCR rxn. 25mM Mg2+ was added as required and is shown on the PCR set up link above.
Results: n00b alert! The gel below is a repeat of yesterday's PCR, except with DNased RNA samples added to verify no gDNA carryover. However, you'll notice that the PCR results on the cDNA don't look identical to yesterday's PCR. Ugh.
Gel Loading (from left to right):
Opsin Primers (top half of gel), Rhodopsin Primers (same loading order, bottom half of gel)
RNA samples (left sides of gel), cDNA samples (right sides of gel)
1 - 100bp ladder
2 - retina
3 - fin
4 - 4th arm
5 - dorsal mantle center
6 - dorsal mantle side
7 - ventral mantle center
8 - ventral mantle side
9 - H2O
Loading is repeated in the above order for the cDNA, which is the right halves of the gel.
Well, the good news is that no signal is seen in the DNased RNA samples, thus confirming that there is no detectable gDNA carryover.
Opsin Primers
We see a band in the retina, fin & ventral mantle center samples as we did yesterday. However, we see a band in the 4th arm sample, which was not present yesterday. We also do NOT see a band in the ventral mantle side sample like we did yesterday. Negative controls are negative.
Rhodopsin Primers
We see a band in the retina and fin samples as we did yesterday. However, we no longer see the dual bands in the ventral mantle center as were seen yesterday. Negative controls are negative.
Bands were excised from the gel, placed in 1.5mL snap cap tubes and stored in the -20C in Sam's "Purified Inserts" box.
Because of the lack of repeated results, I will repeat this PCR for a third time, with just the cDNA again, to see if I can duplicate one set of results...
20091215
PCR - Sepia cDNA (from yesterday)
Set up PCR on recent Sepia cDNA samples using both S. officianalis_rhodopsin_F, R primers & Sep_op_F2, R2 primers.
PCR set up is here. Looking back at my old paper (gasp!) notebook from March 2007, I noticed that each primer set required differing amounts of Mg2+. Rhodopsin primers require 3mM Mg2+ in the PCR rxn and the opsin primers require 2mM Mg2+ in the PCR rxn. Mg2+ was added as required and is shown on the PCR set up link above.
Results:
Gel Loading (from left to right):
Opsin Primers (lanes 2-10), Rhodopsin Primers (same loading order, lanes 12-20)
1 - 100bp ladder
2 - retina
3 - fin
4 - 4th arm
5 - dorsal mantle center
6 - dorsal mantle side
7 - ventral mantle center
8 - ventral mantle side
9 - H2O
10 - H2O
Opsin Primers
We see an intense band in the retina sample, as expected, since opsin is constitutively expressed in this tissue. We also see a band in the fin and both dorsal mantle samples.
Negative controls are blank.
Rhodopsin Primers
We see an intense band in the retina sample. We also see a band in the fin sample. We see two bands in the ventral mantle center tissue, possibly suggesting a rhodopsin isoform is also being expressed in this tissue.
Negative controls are blank.
Overall, these results are rather intriguing because they clearly demonstrate that both genes are being differentially expressed in non-eye tissue of Sepia officianalis.
Reverse Transcription - Abalone 07:12 DNased RNA (from 20090623)
Set up reverse transcription rxns using 174ng of each DNased RNA (sample 07:12-04 was limiting; only 6.1uL available), using Promega oligo dT primers and M-MLV Reverse Transcriptase according to Promega protocol.
RNA/oligo dT primer workup is here. Primer and DNAsed RNA were mixed and brought up to 18.25uL with H2O. Samples were heated @ 70C for 5mins and then placed immediately on ice. The RT Master Mix set up can be found here. 6.75uL of the RT master mix was added to each tube, mixed, spot spun, incubated 42C for 1hr., 95C for 3mins and then the samples were given to Lisa in the Friedman Lab.
Spec - DNased Abalone 07:12 RNA from 20090623
Apparently, these samples had not been spec'd after DNase treatment.
Results:
Samples range in quality (260/280) from not great to perfect. Will perform calcs to make cDNA.
20091214
Reverse Transcription - Sepia DNased RNA (from earlier today)
Set up reverse transcription rxns using 200ng of each DNased RNA, using Promega oligo dT primers and M-MLV Reverse Transcriptase according to Promega protocol.
RNA/Oligo dT primer workup here. Primer and DNAsed RNA were mixed and brought up to 18.25uL with H2O. Samples were heated @ 70C for 5mins and then placed immediately on ice.
The RT master mix set up can be found here. 6.75uL of the RT master mix was added to each tube, mixed, spot spun and then incubated @ 42C for 1hr, heat inactivated @ 95C for 3mins and stored @ 4C.
DNase Treatment - Sepia RNA (from 20091204)
Samples were DNase treated with Ambion's Turbo DNA-free kit, following the rigorous protocol. Used 6uL of each sample, brought up to 50uL with H2O, added 5uL of 10x buffer and 1.5uL of DNase. Incubated 37C for 30mins, added an additional 1uL of DNase and incubated @ 37C for 30mins. Added 0.2 volumes of DNase Inactivation reagent and incubated at RT for 2mins with regular mixing. Spec'd RNA.
Results:
20091211
Bioanalyzer - Herring Liver cDNA for SOLiD Libraries
Samples were run on the DNA 1000 chip for cDNA smear analysis.
Results: 2 of the 4 samples (2L & 3L) look perfect (<20% of cDNA in the 25-150bp range). 6L has <20% of the cDNA in the 25-150bp range (which is perfect), but exhibits a "smear" from ~250-500bp. cDNA in this range suggests overamplification, which will skew gene expression profile. Can repeat PCR for 6L using outer gel slices and reduce the number of cycles to prevent overamplification, if desired. Spoke to Steven and since these samples won't be used to evaluate gene expression (they're for SNP discovery), we won't worry about it for the time being.
Sample 4L has some very strange signals being generated in the ~500-800bp range. Additionally, the virtual gel image (not shown) shows a great deal of smearing, unlike the other samples.
20091209
Emulsion PCR - Herring Liver cDNA for SOLiD Libraries
Emulsion PCR was performed with the two inner gel bands cut out earlier today according to the Ambio WTK protocol. PCR was run for 15 cycles. After the PCR, the samples were cleaned up using the Invitrogen PureLink PCR Micro Kit, according to the Ambion WTK protocol. The cleaned up cDNA (referred to as "libraries" from now on) was spec'd prior to running on the Bioanalyzer.
Results:
All samples look good EXCEPT for 4L. It has a terrible 260/230 ratio and has a very low concentration, relative to the other three samples. Although not pictured here, the absorbance curve of the 4L sample was extremely poor and broad, unlike the other three samples.
Reverse Transcription - Herring Liver mRNA for SOLiD Libraries
Ligation reactions from yesterday were subject to reverse transcription according to protocol.
Master mix workup info is here. Samples were incubated @ 42C, 30mins and then cleaned up using the Qiagen MiniElute PCR Purification Kit, according to the Ambion WTK protocol.
After RT rxn, samples were run on a Novex 6% TBE-Urea gel according to Ambion WTK protocol. Samples were loaded, left to right: 2L, 3L, 4L, 6L. Ladders are to the left of each sample. The break in the smear in the 6L sample is a tear in the gel.
The recommended range of cDNA (100-200bp) were excised from the gel and were cut into four pieces, according to the Ambion WTK protocol. The two outer gel slices from each sample will be stored @-20C. Then proceeded to the emulsion PCR. Here is an image of the gel after the cDNA was cut out:
20091208
RNA Adapter Hybridization and Ligation - Herring Liver mRNA for SOLiD Libraries
RNA from yesterday was speedvac'd to dryness and resuspended in 3uL of nuclease-free H2O. Samples were mixed with Adaptor Mix A and hybridized according to Ambion WTK protocol. Samples were then ligated for 16hrs @ 16C, according to Ambion WTK protocol.
20091207
RNA Fragmentation - Herring Liver mRNA for SOLiD Libraries
Samples from
20091203. 0.5uL was removed from each and transferred to separate tubes and diluted to < 5ng/uL for subsequent Bioanalyzer analysis using the Pico chip. Samples were fragmented using RNase III according to the Ambion WTK protocol and then cleaned up/concentrated using the Invitrogen RiboMinus Concentration Module according to the Ambion WTK protocol.
Samples were spec'd prior to running on the Bioanalyzer:
Concentrations/absorbance values are not accurate when using the NanaDrop after using the RiboMinus Concentration module, according to the Ambion WTK protocol. However, yields seem pretty good...
Total, mRNA and fragmented mRNA from each of the four samples was run on the Pico chip with the Eukaryote Total RNA Bioanalyzer protocol.
Results:
The 2L tot (total RNA) and 3L tot (total RNA) samples are clearly very good quality. 2L tot does exhibit some very slight degradation, though. 4L tot (total RNA) and 6L tot (total RNA) show a much greater degree of degradation. All mRNA samples show complete removal of any trace, contaminating rRNA. The fragmented samples (the last four samples on the gel image above) all appear to be perfect. The 4L frag sample simply has less RNA loaded and that is why it is not as dark as the other three fragmented samples. Despite the degradation in the 4L tot and 6L tot samples, the fragmentation profile looks good and we will proceed with making the cDNA libraries for those samples.
20091204
RNA Isolation - Sepia samples
Isolated RNA from 7 sepia samples received 20091125. Samples were removed from RNA Later, blotted and homogenized in 500uL of TriReagent. 500uL of additional TriReagent was added to the tubes after homogenization. Procedure was followed normally. The sepia retina RNA was isolated separately from the other samples and was resuspended in 100uL of 0.1% DEPC-H2O. The remaining samples were isolated and resuspended in 20uL of 0.1% DEPC-H2O. Nearly all samples had some sort of purple tint to them, ranging from almost black to extremely faint purple hue. The samples were spec'd and then stored @ -80C in Sam's RNA Box #1.
Results:
Nearly all of the samples exhibited very strange curves and mediocre 260/280 ratios (for RNA). Could be due to the "purple stuff" carryover or possibly an effect of the RNA Later from which the samples were stored.
20091203
Sequencing - Dungan Isolates, Lake Trout HRM and Emma DD cloning
Submitted 1.5 plates for Sanger sequencing. Dungan isolates prepared by me, Lake Trout HRM prepared by Rony and Emma's differential display cloning samples prepared by her. All primers were prepped by me. See the sequencing log for samples and plate layout.
mRNA Precipitation - Herring Liver mRNA for SOLiD Libraries (continued from yesterday)
Spun samples 16,000g, 30mins, 4C. Discarded supe, quick spun tubes, removed residual supe, washed with 1mL 70% EtOH. Spun samples 16,000g, 15mins, 4C. Discarded supe, quick spun tubes, removed residual supe, resuspended in 8.5uL of nuclease-free H2O. Stored @ -80C until ready to proceed with fragmentation for SOLiD libraries.
20091202
Hard Clam Challenge - QPX Strain S-1 (continued from yesterday)
All clams appeared to be alive and well. Most had their siphons out when I arrived to start collecting tissues. Clams were shucked after 24hr challenge. Gill and mantle samples were collected in separate 1.5mL snap cap tubes, stored briefly on ice and transferred to -80C in "Hard Clam QPX Challenge 12/2/2009." box.
mRNA Precipitation - Herring Liver mRNA for SOLiD Libraries (continued from earlier today)
Precipitation of mRNA from earlier today in preparation for fragmentation. Fragmentation requires mRNA volumes of <8uL, so after precipitation I will resuspend pellets in 8uL of 0.1% DEPC-H2O. Will use 1ug (this means 1/2 of 3L and 2L & all of 6L and 4L) of mRNA from each sample for precipitation. Remainder of 3L and 2L samples were stored @ -80C in "Herring RNA Box #1."Samples were precipitated by adding 0.1 volumes 5M ammonium acetate, 1uL glycogen and 2 volumes of 100% EtOH. Samples were incubated O/N @ -20C.
mRNA Isolation - Herring Liver RNA for SOLiD Libraries (continued from earlier today)
Clean RNA from earlier today was processed according to the Ambion Micro PolyA Purist Kit to isolate mRNA. This procedure was done two times to ensure full mRNA enrichment of the samples. Samples were then spec'd.
Results:
Yields:
3L - 10.66 ng/uL x 200uL = 2.132ug
6L - 6.97 ng/uL x 200uL = 1.394ug
2L - 10.89 ng/uL x 200uL = 2.178ug
4L - 6.43 ng/uL x 200uL = 1.286ug
The 260/280 for 3L is not that great. The remainder are pretty good, but not superb.
RNA Precipitation - Herring Liver RNA for SOLiD Libraries (continued from yesterday)
Samples were pelleted for 30mins, 16,000g @ 4C. Supe was discarded, samples quick spun, residual supe removed and then washed with 1mL 70% EtOH. Tubes were vortexed until pellet came off of bottom of tube and then spun 15mins, 16,000g @ 4C. Supe was discarded, samples quick spun, residual supe removed and then resuspended in 250uL of 0.1% DEPC-H2O in preparation for mRNA isolation using the Ambion Micro PolyA Purist Kit.
20091201
RNA Precipitation - Herring Liver RNA for SOLiD Libraries
50uL of RNA from each of the following were precipitated O/N @ -20C:
2L HKOD09 - 2.157 ug/uL x 50uL = 107.85ug
4L HTOG09 - 2.089 ug/uL x 50uL = 104.45ug
3L HSITK09 - 1.089 ug/uL x 50uL = 54.45ug
6L HPWS09 - 1.703 ug/uL x 50uL = 85.15ug
0.1 volumes (5uL) of 3M NaOAc ph = 5.2 was added to each sample. Then 2 volumes (110uL) of 100% EtOH was added. Samples were vortexed and incubated O/N @ -20C.
Hard Clam Challenge - QPX Strain S-1
Challenged 2 FL hard clams and 1 BX hard clam with ~100uL of unwashed, 11 day old cultures. 2 FL hard clams and 1 BX hard clam received ~100uL of QPX media, as controls. Injections were done through the hinge using 20G1 needles and aimed for the pericardial cavity. After injections, clams were left out of water for 1.5hrs, then return to small containers of sea water. They will be incubated for 24hrs.
MBL Shipment - Hard Clam gill tissue in RNA Later
Received samples from Scott Lindell today. Two ziplock bags taped together labeled "11/16/09 Clams scudders." The bags contain 2mL screw cap tubes with small tissue samples in RNA later. One group of tubes is labeled with FL-3 # and the other group with BX-4 #. Samples will be stored at 4C to be processed later this month.
20091125
Sea water
Received ~130 gallons of sea water. Water was transferred to the cold room (15C) in to three 55 gal Rubbermaid bins and covered.
MBL Shipment - Sepia tissue samples
Received sepia tissue samples in RNA Later from Kendra Buresch. Here's the list of tissue we received,
according to the Post-It with the samples:
1. 4th arm
2. ventral mantle center
3. ventral mantle side
4. fin
5. dorsal mantle center
6. dorsal mantle side
7. retina x 2
Samples were temporarily stored @ 4C. Will discuss with Steven on long term storage (if necessary).
20091124
MBL Shipment - Hard Clams
Received hard clams from Scott Lindell today. Two bags. One group (4 live clams, 1 empty shell) labeled as "FL" and another group (9 live clams) labeled as "BX." Clams were transferred to separate plastic shoebox containers with sea water and sand. They were stored at 15C until ready for experiment.
20091120
QPX Split
Split off extra QPX according to protocol for upcoming Hard Clam challenge.
20091119
Herring 454 Data
Data from MoGene was received today on two DVDs and one HDD. Data is two runs of two libraries, due to MoGene concerns that the data of the first run looked bad (too few reads). They performed a second run at no charge and provided us with that data as well.
MBL Shipment - MV oysters/cod
Received a shipment of various MV oysters/cod samples from Scott Lindell at MBL. However, these were NOT shipped on dry ice! Samples were put @ -80C. Will be organized at a later date.
20091118
PCR - "Unknown" Dungans/Lyons
This is a repeat of yesterday's set up with LABY primers, but with an annealing temp of 53C in hopes of improving the number of amplicons generated from additional samples. See yesterday's PCR run for info on samples.
Results: Samples were loaded 1-29 and three negative controls from left to right, top to bottom.
The lower annealing temperature clearly resulted in more products. The ~500bp band was cut from each lane and stored @ -20C. All bands will be purified using Millipore sping columns and then sent for sequencing.
20091117
PCR - "Unkown" Dungans/Lyons
This was done on the numbered tubes using the LABY A/Y primers for eventual sequencing. Turns out many of the tubes have some info (other than just a number) on their sides which might provide more information regarding which isolate they actually are.
PCR set up is here. Annealing temp 55C.
Tube-#
|
Side Label
|
1
|
VA1423-1
|
2
|
VA1423 2CB
|
3
|
VA1423-3
|
4
|
VA-1423 4
|
5
|
VA1423-6 6
|
6
|
VA1423-10
|
7
|
VA1423-12
|
8
|
VA1423-15
|
9
|
VA1423-26
|
10
|
VA1423-28
|
11
|
VA1423-290 2003 Isolate
|
12
|
VA1423 29 2004 Isolate
|
13
|
VA-1423-33
|
14
|
VA1423-37
|
15
|
XMAC13T
|
16
|
X-MAC-19T
|
17
|
XMAC 20T
|
18
|
X-MAD 10T
|
19
|
X-MAD-14T
|
20
|
X-MAD-18T
|
21
|
XMAE 11T
|
22
|
XMAE 13T
|
23
|
BC05CA8T
|
24
|
BC05CA 15T
|
25
|
BC05CA 18T
|
26
|
BC05CA 20T
|
27
|
98 MFS 61A
|
28
|
CRT W 1HE/H11
|
29
|
CRSH 5B3
|
Results: Samples were loaded 1-29 and three negative controls from left to right, top to bottom.
There are four prominent bands from Tubes 23, 27, 28, 29. These four bands were excised and purified with Millipore spin columns according to protocol. They will be sent for sequencing. There are faint bands visible from Tubes 9 & 11. Due to the faintness, they were not excised as there may not be enough product for sequencing. The remainder of the samples failed to produce any amplicons.
20091113
Hard Clams - Shipment from Rutgers
Received Hard Clam gill samples on "wet ice" in RNA Later from Rutgers. Samples were collected on 11/4/09 (clams held in refrigerator) and preserved (gill tissue collected) on 11/9/09
according to the paper included with the samples. Samples will be stored @ -80C until we are ready to process.
20091110
Sequencing - Lake Trout HRM
This is a second submission of 12 individuals from 8 primer sets. The previous sequencing run was botched because I used the combined primer plate instead of a single (forward or reverse) primer for submission.
20091105
Oyster CO2/Mechanical Stress - Water quality
See
Rachel's 441 Notebook from 10/28/2009 through 11/4/2009 for experiment info. 500mL of water was collected from the CO2 and the air tanks. Water was vacuum filtered through Watman paper. The Watman paper was allowed to dry over the weekend.
Results:
CO2 = 35.9mg
Air = 14.4mg