Secondary stress: heat shock wrap-up
Sam and Lisa attended to the heat shocked oysters while I was gone this past week.
Dec 21: none dead
Dec 22: all dead in light blue (43°C heat shock), 1 dead in purple (42°C)
Dec 23: none dead
Dec 24: 2 dead in purple, high ammonia and low pH so Lisa did a 50% water change
Dec 25: none dead, high ammonia 50% water change
Dec 26: none dead
Dec 27: end experiment
100% mortality from 43°C heat shock by 72 hours post-shock. 30% mortality from 42°C heat shock by 96 hours post-shock.
December 20, 2011
Secondary stress
Checked the oysters around 11 am and 1 was dead in the 43°C heat shocked group (light blue zip tie). Decreased water level and fed oysters 2 mL of shellfish diet diluted in 300 mL of seawater. After 1 hour, the water was not clear (oysters were not eating much).
December 19, 2011
Secondary stress
Began heat shock of oysters to find LHT at 12:15 pm. 10 oysters were used for each temperature. Beakers were filled to 800 mL with seawater and placed in the water bath and allowed to equilibrate to temperature. Prior to 1 hour long heat shock, oysters were preheated for 10 minutes in a separate beaker. The preheating is done to make sure that the internal temperature of the oysters actually reaches the temperature of the water and to avoid the initial decrease in T that occurs when the oysters are put in the beakers.
| Time |
HeatShockTemp |
Color |
| 12:15 |
38°C |
green |
| 13:47 |
39 |
darkblue |
| 15: 36 |
40 |
red |
| 17:45 |
41 |
brown |
| 19:15 |
42 |
purple |
| 20:40 |
43 |
lightblue |
Checked the ammonia of the tank around 4 pm. Was at about 0.25 ppm. Added more denitrifying bacteria and did partial water change.
OA FHL 2011
qPCR same as EF1a of FHL OA samples on 12/15/11 but using v-type H+ transporter primers with an annealing T of 60°C.
Results: All cDNA samples amplified but had double melt peak. There was no amplification in gDNA or NTCs. There was no difference in normalized expression among treatments.
December 18, 2011
Secondary stress
Fed the oysters at 3:30 pm by diluting 2 mL of shellfish diet in about 300 mL of seawater, reducing the water level in the tank, and letting the oysters feed for 1 hour.
The ammonia was between 0.25-0.5 ppm so did a partial water change.
December 17, 2011
Secondary stress
Fed the oysters around 9 am but only put 2 mL of shellfish diet in the water. They cleared all algae after about 1 hour. Temperature was about 13.6°C and ammonia was 0.5 ppm. Did a complete water change including rinsing out sediment/pseudofeces at the bottom of the tank. Added more denitrifying bacteria after the water change.
OA FHL 2011
qPCR using v-type H+ transporter primers, exact same PCR as yesterday except used 60°C annealing T.
Results: gDNA did not amplify (neither did NTCs) and cDNA did. However, there is still a double melt peak for the cDNA.
qPCR using v-type H+ transporter primers, only cDNA and NTCs. Annealing Ts of 61 and 62°C.
Results: Similar to annealing of 60°C - cDNA amplified but there was a double melt peak. Amplification was significantly decreased at 62°C indicating the efficiency of the reaction is decreasing at that T (which can also be seen on the gradient gel using the same primers, the PCR product at 62.7°C is much more faint than that at 61.1°C).
December 16, 2011
Secondary stress
Picked up 77 oysters from Oyster Bay in Shelton (Taylor Shellfish) that were harvested on the midnight tide. Brought them to the basement and put them in the tank around 10 am and set the temperature at 55°F (~13°C). The ammonia was at 0.1 ppm in the tank, so added ~1 cup more of denitrifying bacteria.
Details on oysters:
spawn #6 from 2011, Quilcene Hatchery, raised in flub AF
Planted on tide flats of Oyster Bay 8/31/2011
Parents were from Molluscan Broodstock Program group "G", a combination of 3 families: 20.010, 20.019, and 20.037
At 2 pm checked on the oysters. The temperature was about 13.6°C in the tank. Some of the oysters were obviously filtering water. The ammonia was at about 0.5 ppm. Did a partial water change.
At 5 pm lowered the water in the tank and fed the oysters by diluting 4 mL of Shellfish Diet 1800 in seawater. This seemed like a lot of algae and after 30 minutes the oysters had not noticeably cleared the water. The ammonia was only about 0.1 ppm. Put the water back in the tank. Temp was about 13.6°C.
OA FHL 2011
qPCR of all cDNA samples in duplicate using glutamine synthetase primers, including gDNA and NTCs.
Results: All samples amplified except for 105A2 and 103B3. There was no amplification in the gDNA controls or NTCs. ANOVA of the normalized gene expression shows no significant difference between treatments.
qPCR of all cDNA samples in duplicate using aspartate aminotransferase primers, including gDNA and NTCs. The plate had the exact same layout at the glutamine synthetase. Annealing T for aspartate aminotransferase was 63.5°C.
Results: cDNA amplified except for 1 of the replicates each for both 105A2 and 103B3. The replicates that did amplify for those samples came up at >39 Cq. Analysis based on PCR miner, which did not detect amplification in either of those samples, finds no difference between treatments.
qPCR of cDNA, gDNA and NTCs using v-type H+ transport primers with annealing T of 56°C.
Results: cDNA replicates amplified well and in concordance with each other. The melt peak has 2 peaks, though. The 3 gDNA samples amplified (no amp in NTCs) with funky melt peaks.
December 15, 2011
OA FHL 2011: primers
Gradient PCR using new primers: v-type H+ transporter. Master mix is made of 12.5ul Apex, 10.5 ul water, 0.5 ul of each 10 uM primer, and 1 ul template (cDNA or gDNA). cDNA is in the first row of the plate (with a NTC in well 2) and gDNA is in the second row. The PCR protocol is 95C for 10 min, 40 cycles of 95C 15s, annealing T 15s, and 72C 30s, followed by 95C 10s and 72C 10 min. The annealing T range from 50-65C: 50, 50.4, 51.3, 52.5, 54.2, 56.4, 59, 61.1, 62.7, 63.8, 64.7, 65.
Results: cDNA amplified well through annealing T of 59°C. gDNA shows non-specific binding through an annealing T of 54°C (with primer dimer still apparent at 56°C). qPCR at annealing T greater than or equal to 55°C should work well for cDNA-specific binding.
OA FHL 2011
qPCR using EF1a primers of FHL OA samples in duplicate , 3 ul of template per reaction. 2 gDNA controls and 3 NTCs on the plate.
Results: All cDNA samples amplified and replicates came up at the same Cq. All amplified products had the same melt peak. There was no amplification in any of the NTCs nor in the gDNA controls.
qPCR using glutamine synthetase and aspartate aminotransferase primers at previously determined annealing Ts of 67.5 and 63.5C, respectively. Included gDNA and NTCs on the plate.
Results: The dropbox on the computer malfunctioned and I am not able to export the data or report, but both primer pairs amplified a single product with replicates in good agreement for cDNA. There was no amplification in gDNA or NTCs.
Secondary Stress
Sammi helped me set up a large tank with filters and water chiller for the oysters. We set the temperature at 59°F. Added ~2 cups denitrifying bacteria (Proline).
December 12, 2011
OA FHL 2011: primers
SR aligned mRNA for v-type H+ transporters (see 12.9.11) with genomic sequence from BGI for C. gigas to determine intron/exon boundaries. See his lab notebook for details. I designed primers to span the intron/exon boundaries at base pairs 1419 (1410-1429) and 1592 (1585-1604). The reverse primer was designed off of the reverse complement of the sequence. The primers are both 45% GC with Tm of 49.73°C. The SRIDs are 1436 and 1437.
OA FHL 2011: reverse transcription
Reverse transcribed remaining RNA from July 29, 2011 C. gigas larvae. Since we're not sure what the true concentration of RNA (vs. gDNA) in the samples is, it seemed the safest to reverse transcribe all of it to get as much cDNA as possible. For each volume of RNA, scaled up the amount of MMLV reagents used based on a total volume of 17.75 µL RNA for 1 reaction. The master mix in the table refers to 5x MMLV RT buffer (5µL per reaction), 10 µM dNTPs (1.25 µL), and 0.5 µL reverse transcriptase.
| Sample |
VolumeRNA |
VolOligodT |
VolMasterMix |
| 105A1 |
30 |
0.85 |
11.41 |
| 105A2 |
22 |
0.62 |
8.37 |
| 103A3 |
26 |
0.73 |
9.89 |
| 103A4 |
30 |
0.85 |
11.41 |
| 103B3 |
32 |
0.90 |
12.17 |
| 103B5 |
29 |
0.82 |
11.03 |
qPCR of samples with greatest volume of cDNA: 105A1, 103A4, and 104B3. qPCR used 2 µL of undiluted cDNA with NOAA OA 2000A cDNA as a positive control and 3 NTCs. Primers were EF1a at annealing T of 66°C.
Results: The positive control and all 3 samples amplified and had very similar melt peaks. The FHL OA cDNA samples all amplified by ~cycle 35, but more template may be needed to a) increase the efficiency of the reaction and b) make sure that expression of other genes is detected.
December 9, 2011
OA FHL 2011: primers
Primer design for another OA-response gene. Mined results from Todgham & Hofmann 2009 and Stumpp et al. 2011 as well as larval clam response to OA to find candidate genes. All three showed that H+ transporters are differentially regulated upon OA exposure. Searched Sigenae BioMart using GO term 0006818 (proton transport) and got 84 contigs in return. A number of them correspond to v-type proton ATPase, which is also a transporter that Portner indicated would be important in invertebrate response to OA. The C. gigas accession numbers that correspond to this gene are:
| AM854014 |
AM854014 |
AM855635 |
AM858421 |
|
| AM864981 |
||||
| AM868052 |
CB617449 |
|||
| CU681539 |
||||
| CU682217 |
Did exact same EF1a qPCR as 12.7.11 but used new primer stocks ordered to replace the possibly contaminated ones.
Results: All cDNAs at all 3 annealing Ts amplified with replicates at each T being identical. Only one gDNA (at 65°C) amplified, but it was probably not the gDNA itself. About half of the NTCs showed a low level of contamination. After a conference with Steven and Sam, we're not really sure what is going on. The cDNA amplifies very well despite whatever is going on in the NTCs. It's unlikely that the NTCs are contamination since the amplification is not consistent. (Amplification plot is log scale.)
qPCR using EF1a primers and just NTCs (x3).
Results: A small amount of amplification occurred in each NTC.
December 7, 2011
OA FHL 2011: primers
qPCR of cDNA, gDNA and 3 NTCs with EF1a primers at annealing Ts of 67.5, 66, and 65°C.
Results: cDNA amplified at all annealing temperatures. All annealing temperatures demonstrated identical melt peaks that seem to be a high quality product (results from 12/6/11 were probably just a bad reaction for some reason).
67.5°C - all NTCs were clean and no amplification occurred in the gDNA samples.
66°C - 2 NTCs were clean and 1 gDNA sample amplified
65°C - 1 NTC was clean and 1 gDNA sample amplified
The unlabeled amplification curves and melt peaks correspond to the gDNA and NTC samples that amplified.
qPCR of cDNA, gDNA (x3) and 3 NTCs with aspartate aminotransferase primers (AY660003) with annealing T of 63.5°C.
Results: Only the cDNA amplified - there was no amplification at all in the gDNA or NTCs. The product showed a single melt peak.
December 6, 2011
OA FHL 2011: primers
Gradient PCR with same conditions as 11/18 except used just 1 µL of template. After PCR, products were run on 1.5% agarose gel + EtBr with Hyperladder II (Bioline).
Results: Annealing temperatures are in blue at the top of the wells. The primers amplified a product of about 100 bp in the cDNA and amplification was strong through annealing temperature of 67.7°C. There was a lot of non-specific binding in the gDNA, but the amplification got considerably weaker after 57.5°C and was almost non-existent by 66.1°C. Next step will be to do a qPCR testing cDNA and gDNA at an annealing temperature of 67.5°C.
qPCR of cDNA, 3 gDNA replicates and 3 NTCs using EF1a primers with an annealing T of 67.5°C.
Results: the cDNA amplified, but the melt peak was weird. There was no amplification in the gDNA or NTCs. Will try qPCR again at the same annealing T as well as annealings of 66 and 65°C.
December 5, 2011
OA FHL 2011: primers
qPCR to check validity of using EF1a primers in selectively amplifying cDNA. One sample of cDNA as positive control and 3 samples each of gDNA and NTC.
Thermalcylcer protocol is 2stempamp+melt EvaGreen (55°C annealing T).
Results: cDNA amplified well. 1 NTC was clean but the other 2 had amplification (same melt peak as the cDNA). gDNA amplified a little bit, but had a very different melt peak than cDNA. Next step is to do a gradient to see if amplification of gDNA can be gotten rid of (since it is probably non-specific binding). Will also bleach down lab bench and make new primer stocks before next qPCR to try to get rid of contamination.
Limit of detection
qPCR of samples reverse transcribed 12/1/11 using EF1a primers (SRID 1410 and 1412). Thermalcylcer protocol is 2stempamp+melt EvaGreen (55°C annealing T).
Results: One NTC had some contamination, but it amplified much later than the samples. All samples amplified between Cqs of 26-34.
December 1, 2011
OA FHL 2011
Ran gradient PCR using new aspartate aminotransferase primers (SRID 1422 and 1423). The Gradient ran from 50-65°C using the Opticon. Mastermix was made of 12.5 µL Apex, 9.5 µL water, and 0.5 µL each of 10 µM primers with 2 µL cDNA (Gill C9) or gDNA (Gill 0627) template. Thermocycler profile was 95°C 10 min; 40 cycles of 95° 15s, annealing T 15s, 72° 30s; 95° 10s 72° 10 min.
Results: It looks like 63.8°C may be a good annealing temperature to selectively amplify the cDNA and not the gDNA amplicon.
qPCR using AY660003 primers of gDNA (Gill 0627) and 2 larval cDNA (NOAA OA Sept 2010 380A and 2000A). Annealing temperature of 63.5°C.
Results: Win! At an annealing T of 63.5°C the primers do not amplify gDNA but do amplify the larval cDNA. Good job, Steven!
Reverse transcription - limit of detection
Reverse transcribed DNased larval cDNA from NOAA OA Sept 2010, samples 280C and 2000A. For each sample, did RT of 0.05, 0.1 and 0.3 µg (See below for volumes). For each volume x of RNA, added 17.75-x µL of water and 0.5 µL Oligo dTs. Incubated at 70°C for 5 minutes. Put on ice for a few minutes then added 5 µL MMLV 5x buffer, 1.25 µL 10 mM dNTPs and 0.5 µL reverse transcriptase. Incubated at 42°C for 1 hour followed by 3 minutes of deactivation at 95°C.
November 28, 2011
OA FHL 2011
Ran gel (1.5% agarose + EtBr) of PCR done 11/23/11. gDNA amplified with a product of about 550 bp and the cDNA amplified a product less than 200 bp. There was no primer dimer and the NTCs were clean.
Future steps: Try increasing the annealing temperature of the primers (originally amplified at 50°C).
qPCR using primers that amplify only cDNA (EF1a) and primers that amplify both cDNA and gDNA (18s). Samples used: Gill C9 (positive control cDNA) and newly reverse transcribed FHL OA 105B4 and 105B6.
Results: Both primer pairs amplified C9, the positive control. 105B6 amplified a small amount for EF1a (pink) and 18s (blue), although there was some contamination in the 18s NTC. 105B4 appears to have not amplified at all.
November 23, 2011
OA FHL 2011
Reverse transcribed Dnased RNA samples 105B4 and 105B6. The cDNA from these samples will be used as test runs for the new primers. To 17.75 µL of RNA, added 0.5 µL Oligo dTs and incubated at 70°C for 5 minutes. Put samples on ice for about 3 minutes then added to each 5 µL of MMLV 5x buffer, 1.25 µL of 10 mM dNTPs, and 0.5 µL of reverse transcriptase. Incubated at 42°C for 1 hour followed by a 95°C inactivation for 3 minutes.
qPCR of new cDNA with EF1a primers and glutamine synthetase primers. Also included Gill C9 as a positive control and 2 NTCs. GS was amplified at 67.7°C annealing T and EF1a at 55.
Results: NTCs were clean and C9 amplified for both primer sets. None of the larval cDNA amplified.
qPCR of NOAA OA 2000 ppm cDNA and Gill C9 with EF1a primers to make sure the primers work with larval cDNA (this cDNA has been successfully amplified with multiple primers in the past).
Results: The EF1a primers successfully amplified both adult and larval cDNA. The NTCs were clean.
Received the primers that SR designed for aspartate aminotransferase (Cg_AY660003_F SRID 1422 and R SRID 1423). Did regular PCR using Apex (12.5µL), water (8.5 µL) and 10 µM primers (0.5 µL each) with an annealing temperature of 50°C. Layout of samples in strip tube for PCR is Gill 0627 gDNA (A), gDNA (B), Gill C9 cDNA (C), cDNA (D), NTC (E and F).
November 21, 2011
OA FHL 2011
qPCR with glutamine synthetase (annealing T of 67.7°C) and Tld (66.1°C) using gDNA and cDNA.
Results: gDNA did not amplify for either primer pair at the indicated annealing temperatures and cDNA did! NTCs were clean for GS, but there is evidence of primer dimer in the Tld. This may not be a problem if the primers bind only to the PCR product when it is present. However, there is primer dimer below the cDNA PCR product for Tld (11/18/11) at this annealing T so it may be worth it to decrease the primer concentration in the reaction.
qPCR with glutamine synthetase primers at new annealing temperature with cDNA (used originally for preliminary data of SOD, GPx, Prx6 for FHL OA) diluted 1:5 in nanopure water, in duplicate.
Results: Nothing amplified :( But at least the NTCs were clean!
qPCR with glutamine synthetase primers with undiluted cDNA from FHL OA and positive control cDNA from Gill C9.
Results: Gill C9 amplified with a single melt peak. The FHL OA cDNA did not amplify - probably need more template or this gene is not expressed in these samples.
November 18, 2011
OA FHL 2011: primers
Ran a gradient PCR on the Opticon from 55-70°C for primers that showed evidence of non-specific binding in gDNA and amplified cDNA well. If the annealing temperature can be optimized so that the non-specific binding ceases in the gDNA but specific binding continues in the cDNA, then the primers will work. The primers that fit these criteria are: ATP synthase, Prx6, glutamine synthetase, Tld, and citrate synthase. All primers were run in this PCR except for the citrate synthase. No PCR was done for 55.4 or 70°C since they were redundant with other annealing temperatures. Negative controls for each primer pair were PCR'd at 55.4°C.
Results: All gDNA and cDNA amplified at 55°C and 56.3°C. Beginning at 57.5°C, the gDNA non-specific binding for glutamine synthetase (GS) and Tld began to noticeably decrease and stayed decreased, although looked like it never completely disappeared. Annealing continued for gDNA of ATP synthase and Prx6 throughout the gradient. Binding of cDNA failed for ATP and Tld at 67.7°C. The only gene (cDNA) that the primers annealed to at 68.8°C was with the GS primers and there was still a faint amount of gDNA amplified at a much larger amplicon size. Primer dimer was apparent in all 4 NTCs (at the very end of the 3rd row of the gel).
Future steps: It's worth trying qPCR for Tld and GS at higher annealing temperatures, but the primer binding for the other genes is probably not non-specific and the primers will need to be redesigned.
November 17, 2011
OA FHL 2011: primers
Ran PCR from 11/16/11 and the IMSP-5 PCR reactions from 11/14/11 on a 1.5% agarose gel with EtBr at 100 V for 45 minutes. The gel drifted during the electrophoresis.
Results: IMSP-5 showed a lot of non-specific binding in the gDNA and no amplification in the cDNA. Aspartate aminotransferase amplified two products in the gDNA - one the same size as the cDNA amplicon and one much larger. The MTIV primers didn't amplify anything. The phosphoglucomutase primers amplified the same product size in gDNA and cDNA, implying that there is no intron in the gDNA at this site. All NTCs were clean.
November 16, 2011
OA FHL 2011: primers
Received the new (R2) reverse primers for aspartate aminotransferase (SRID ), MTIV ( ), and phosphoglucomutase ( ) that are the reverse complement of the previous reverse primers. Did PCR as described 11/14/11.
November 14, 2011
OA FHL 2011: primers
Did PCR using primers designed to not amplify gDNA: Tld, glutamine synthetase (GS), hsc70, ATP synthase, Prx6, citrate synthase, and IMSP-5. Each PCR reaction had 12.5 µL Apex master mix, 8.5 µL water, 0.5 µL of each primer, and 3 µL of template (either gDNA Gill 0627, cDNA gill C9, or water for controls). For Tld, GS, and hsc70, the new reverse primers were used. The PCR was run using the QPCR thermalcycler profile in the EMMA directory. 10 µL of the PCR products were run on a 1.5% agarose gel with EtBr alongside 5 µL of Hyperline ladder II. There was not enough room on the gel for the IMSP-5 reactions.
gDNA is marked with a green "g", cDNA with a blue "c" and the negative control lanes are not marked at all.
Results: All negative controls were clean. Tld has a lot of primer dimer. Tld, GS, hsc70, and citrate synthase all show multiple bands (products) amplified in the gDNA. All primers amplified gDNA products that are larger than expected for the cDNA amplicon, suggesting that these genes have introns. hsc70 is the only primer pair that amplified a gDNA product of the same size as the expected cDNA product. All cDNA amplicons are the expected size and only show a single product.
November 11, 2011
Histology practice
At 10:30 am (24 hours after putting the samples in Davidson's) I put the two tissue samples in 70% EtOH.
OA FHL 2011: primers
qPCR using new reverse primers for Tld, glutamine synthetase, and hsc70. All of them amplified both gDNA and cDNA :( Melt peaks were different between gDNA and cDNA for all primer sets.
November 10, 2011
Secondary Stress trial
Took samples from all jars for TA and spec pH at 8 am, then did a complete water change of all jars and took 1 sample from the new water for TA and spec pH. Salinity of the water in the jars was 29.5 and in the new water was 29.2 ppt. pH had dropped in all oyster jars. TA didn't drop much in the jars with 4 oysters in them, but did drop in the jars with 8 oysters.
Amy did 2 titrations and spec pH at noon. She did not record the mass of the water sample, so I cannot calculate TA from the titrations.
Histology practice
Carolyn showed me how to fix samples for histological analysis. We carefully shucked 2 oysters ("Emma 1" and "Emma 2") and slid the whole body onto a kim wipe inside a weigh boat. We cut 3 sections for Emma 1 (gill + heart + kidney; gill + DG; adductor) and 2 for Emma 2 (gill + DG; adductor). The sections need to be cut cleanly (1 cut) and thick enough so that they hold their shape. The side that you are interested in looking at should be put face-down in the small plastic box. The sections were put in Invertebrate Davidson's solution and will be left there for 24 hours.
November 9, 2011
Secondary Stress trial
This 2-day trial is designed to determine if smaller oysters alter the chemistry in 4 L jars as much as the previous batch. 4 4-L jars will be filled with water and 2 will have 4 oysters, the other 2 will have 8. At 3 time points throughout today and tomorrow, pH and TA will be measured in each jar. pH is measured using spec pH and TA is measured by doing a manual titration with 0.1 N HCl. For the titration, ~120 g of seawater is weighed into a clean and dry beaker and its mass is recorded. The mass of the bottle + HCl is also recorded, as is the initial pH, voltage (mV) from the pH electrode, and temperature of the sample. The seawater sample is placed on a stir plate and enough acid is added to bring the pH to between 4 and 3.5. Wait a few minutes to let the sample off-gas and the pH probe to equilibrate. Record the mass of the acid, the pH, the voltage, and the temperature. Titrate the sample using the acid, recording acid mass, pH, voltage, and temperature at each titration point until a pH of 3 has been reached.
At 9:00 am weighed out oysters and put them in the jars in a 59 °F water bath in the basement. Also took samples for pH and TA (one pH sample, 2 TA) and measured salinity with the YSI. Samples for a dye correction curve for the spec pH were also taken and their pH was adjusted with NaOH or HCl. The masses of the oysters are:
4Cg-1 46.4 g
4Cg-2 112.6 g
8Cg-1 134.9 g
8Cg-2 182.1 g
The pH of the water at the start of the experiment (corrected for dye addition) is 7.87. The salinity is 29.2 ppt. The 2 measurements of TA are 943 and 902 µmol/kg of seawater.
Amy took samples for the 1 pm and 5 pm time points.
OA FHL 2011: primers
The primers designed on 10/17/11 were designed incorrectly. Both the forward and reverse were designed on the same RNA/DNA strand. For glutamine synthetase (SRID 1417), hsc70 (SRID 1416), and Tld (SRID 1418), I re-ordered primers that are the reverse complement of the ones previously designed.
November 7, 2011
OA FHL 2011: primers
Regular PCR of aspartate aminotransferase and Tld primers to test for primer dimer. PCR reactions are : 12.5µL Apex, 8.5 µL water, 0.5 µL of each primer, and 3 µL of cDNA (Gill VE1). The thermalcycler protocol is QPCR under the EMMA directory.
Made a 1.5% agarose gel. When the PCR was done, loaded 10 µL of PCR product on the gel (5 µL of Hyperline II ladder went in the first well). The gel layout is as follows : 2 wells of asp. aminotrans. + template, 2 negative control asp. aminotrans, the same pattern for Tld. The Tld reactions mostly evaporated in the thermalcycler, but I tried to reconstitute with 10 µL of water and loaded them anyway. Ran at 100 V for about 45 minutes.
Results: None of the Tld samples showed up. The aspartate aminotransferase has primer dimer (underlined in blue) and a faint product of approximately the correct size (circled in orange) showed up in one of the template wells.
qPCR gradient (50-60 °C) using primers MTIV and glutamine synthetase. Used both cDNA and gDNA as template to see if there is an annealing temperature that will amplify only cDNA. Both primers previously amplified both gDNA and cDNA but had very different melt curves for the two amplicons. The thermalcycler protocol used was a modified 2 step amp eva green+melt (modified to include the gradient).
Results: The products that amplified are marked with stars. All NTCs were clean. The MTIV primers amplified gDNA at all temperatures, but cDNA at only 50-52°C. The glutamine synthetase primers amplified cDNA at 50 and 50.7 °C and gDNA at 50-52°C. The melt peaks for both sets of primers were different between cDNA and gDNA.
NB: I found my box of cDNA sitting on the bench next to the fridge. Not sure how it got there or how long it has been sitting there...
November 4, 2011
Secondary Stress Trial
Ammonia levels were low, but still did a water change for oysters.
November 2, 2011
OA FHL 2011: primers
qPCR of new EF1a primers with the re-ordered reverse primer.
October 31, 2011
Secondary Stress Trial
Water change for oysters.
October 28, 2011
OA FHL 2011: primers
The reverse primer for the EF1a qPCR done on 10/27/11 was the reverse complement of what it should have been. Re-ordered the primer in the correct orientation (Cg_EF1a_R2, SRID 1412). Also designed a forward primer for EF1a so that we can empirically determine if there is an intron where we think there is one (Cg_EF1a_F2, SRID 1413).
October 27, 2011
OA FHL 2011: primers
Tested new EF1a primers and further testing of Tld and citrate synthase, which previously seemed to not amplify gDNA.
Results: All products amplified to some degree. The Tld NTC was contaminated and there was no melt curve with the template (it is looking more and more like Tld is only amplifying primer dimer, not a PCR product). The EF1a melt curves were different between cDNA and gDNA. Citrate synthase had different melt curves between the 2 replicates, which could be an indication of different-sized products (i.e. an intron) in the gDNA.
Secondary Stress Trial
Brent picked up new oysters from Joth for a second go at controlling pH in static containers. The oysters are in a 50 gallon tank in the basement. They will be fed twice daily and water changes will be done with the ammonia levels exceed 0.1 ppm>
October 21, 2011
Secondary Stress Trial
Fed bivalves (see spreadsheet).
From Dave's second heat stress with the clams, 3 clams in the brown bag were dead this morning.
qPCR of normalizing genes to see if they amplify only cDNA (not gDNA). Used the sample templates as 10/20/11 and used all normalizing gene primers that we have: EF1a, GAPDH, Actin, 18s, 28s, ARP.
Results: GAPDH, actin, 18s, 28s and ARP primers amplify both genomic and cDNA. NTC amplified for GADPH and 18s. All products had identical melt curves for cDNA and gDNA, except for ARP. EF1a looks like it may be cDNA specific, but I will do another PCR to check.
Results: cDNA amplified much sooner than any of the gDNA samples, but they still came up late in the cycles. NTCs were clean.
For FISH 441
Used NaOH to bring pH of TE buffer to 8.5. Autoclaved TE buffer and EDTA (made 10/13/11).
October 20, 2011
OA FHL 2011
qPCR of gDNA and cDNA with new primers described 10/17/11. gDNA sample used is from SJW - Gill tissue 0627 - and cDNA is Gill VE1. Thermalcycler protocol is 2 step amp eva green + melt.
Results: Products that amplified are indicated with a "*". cDNA for aspartate aminotransferase, Tld, MTIV, and 18s showed very little amplification. Melt curves between gDNA and cDNA were different for hsc70, aspartate aminotransferase, phosphocomutase, MTIV, and 18s. There were not melt curves for glutamine synthase, Tld, and cDNA for aspartate aminotransferase, phosphoglucomutase, and 18s. There was NTC amplification for aspartate aminotransferase, Tld, and 18s.
Next steps: Check possible primer dimer in aspartate aminotransferase, Tld, and 18s. Tld and glutamine synthetase may work (i.e. may not amplify gDNA), but glut synth. does not amplify anything at this annealing temperature and Tld shows evidence of some contamination. Aspartate aminotransferase probably has primer dimer. All other primers amplify genomic DNA.
For FISH 441
Made 2x SDS reducing buffer (10 mL).
- 2 mL 20% (w/v) SDS
- 2 mL glycerol
- 40 µL 2.5% Bromophenol blue
- 2.5 mL 0.5M Tric HCl, pH 6.8
- 2 mL 2B-mercaptoethanol
- 1.46 mL Nanopure water
October 18-19, 2011
Secondary Stress Trial
Fed bivalves (see spreadsheet).
Rehydrated primers in TE buffer pH 8.5 to 100 µM (primers were designed 10/17/11). Let sit at room temp overnight.
October 17, 2011
Secondary Stress Trial
Fed bivalves (see spreadsheet).
OA FHL 2011
Designed more primers to span the intron boundary to avoid amplifying gDNA. Genes were picked from a list of genes that have genomic sequence and, for the most part, for which intron/exon boundaries are known. Genes were also picked based on functionality: energy metabolism, immune, and housekeeping. Two genes were picked that did not yield usable boundaries for primer design: ATB0 (Accession Number AB185609, an amino acid transporter) and a metallothionein gene promoter (AJ249659). The following are the genes for which I designed primers:
hsc70, heat shock cognate 70 (AJ305315): gDNA sequence was aligned with RNA sequence (HS119891) that was retrieved in a megablast against ESTs, narrowing the search to Crassostrea. The alignment was done in Spidey to find the intron/exon boundaries. The primers were designed on the RNA sequence and meant to span the boundary that occurs at the RNA bp 571/572. The SRIDs for these primers are 1396 and 1397.
glutamine synthetase(AJ564739): This gene is used in metabolism of nitrogen. The intron/exon boundaries are known and the primers were designed off of the gDNA sequence. The forward primer spans the intron from bp 751-876 and the reverse spans the intron from bp 1024-1299.
The SRID for these primers are 1398 and 1399.
aspartate aminotransferase (AY660003): This gene is used in amino acid metabolism and changes expression during environmental stress. The primers were designed off of the RNA sequence after alignment with gDNA in Spidey. The intron/exon boundary occurs at RNA bp 138/139 and the forward primer overlaps it. The forward primer goes from bp 128-147 and the reverse from 249-268. The SRID for these primers are 1400 and 1401.
phosphoglucomutase (AJ512213): This gene is involved in metabolism. The primers were designed on the genomic DNA sequence, for which the intron/exon boundaries are known. The forward primer spans the intron from bp 699-1540 and the reverse is within the exon at bp 1730-1749. The SRID for these primers are 1402 and 1403.
Tolloid-like protein, Tld (AJ543452): Involved in the immune response. The primers were designed on the genomic DNA sequence. The forward primer spans the intron from bp 1424-2335 and the reverse occurs within an exon at bp 3457-3755. The SRID for these primers are 1404 and 1405.
MTIV, metallothionein IV (AM265551): The primers were designed on genomic DNA. The forward primer spans an intron from bp 54-156 and the reverse is within an exon at bp 708-817. The SRID for these primers are 1406 and 1407.
18s (EF035120): Housekeeping gene. The primers were designed on genomic DNA, however the boundaries were found using Spidey. The forward spans an intron from 656-670 and the reverse spans a couple of introns from 813-824. The SRID for these primers are 1408 and 1409.
October 14-16, 2011
Secondary Stress Trial
Fed bivalves (see spreadsheet).
Got new algae on 10/16. Put some of it in f/2 in a flask.
October 13, 2011
Secondary Stress Trial
Fed bivalves in the morning and evening (see spreadsheet).
2 clams from the purple bag (39°C heat shock) were dead in the morning and 3 were dead in the evening.
Made 0.5 M EDTA, pH 8
Dissolved 18.61 g of EDTA in 100 mL of Nanopure water by first dissolving about 1/3 of the EDTA in ~50 mL of water. The pH of this starter solution was increased by adding NaOH pellets until the solution was clear. Then the rest of the EDTA was slowing added, along with more NaOH pellets, all the while constantly stirring. NaOH pellets were slowly added to bring the pH up to 8. More water needed to be added too since the solution at one point got too viscous. When the pH was at 8 and all solute was dissolved, the solution was poured into a graduated cylinder and the entire volume was brought to 100 mL. The solution was then put back on the stir plate and brought to pH 8 again using a few drops of 10 N HCl.
Made 20% SDS
Dissolved 20 g of SDS in 100 mL of Nanopure water. First put beaker on stir plate with ~ 50 mL of water, then slowly added SDS. Stirring was slow to avoid sudsing. When the SDS was no longer going into solution, turned heat on low and continued adding. Added a little more water to help with dissolution. After all SDS was added and dissolved, poured solution into a graduated cylinder and brought to 100 mL.
October 12, 2011
Secondary Stress Trial
Fed bivalves in the morning (see spreadsheet).
No mortality from any of the heat treatments.
October 11, 2011
Secondary Stress Trial
Fed bivalves in the morning (see spreadsheet).
No mortality from any of the heat treatments.
October 10, 2011
Secondary Stress Trial
Heat shock of clams and oysters to find lethal heat treatment (LHT). Water bath is brought to temperature and beakers of salt water are placed in the bath to also equilibrate. Once the beakers are at the correct T (checked with a thermometer), oysters/clams in mesh bags are placed in the beaker. They heat shock lasts 1 hour, at which point the animals are removed and (still in the bag) placed into the 50 gallon acclimation tank in the basement. Different heat treatments are indicated by differently colored zip ties on the oyster/clam bags. Below is a table of zip tie colors for the heat shock temperatures (HSTemp).
| HSTemp(°C) |
30 |
33 |
36 |
39 |
40 |
41 |
42 |
43 |
45 |
| Clams |
brown |
darkblue |
lightblue |
purple |
Green |
||||
| Oysters |
dark blue |
Light blue |
Red |
Green |
brown |
The bivalves were not fed this pm.
October 8-9, 2011
Secondary Stress Trial
Fed bivalves twice on Saturday and Sunday (see spreadsheet).
October 7, 2011
Secondary Stress Trial
Fed bivalves (see spreadsheet).
Weighed 5 clams and 5 oysters (total weight). Then shucked them and weighed all the clam shells together and all the oyster shells (shell weight). Total weight - shell weight will give an estimate of wet weight to possibly help explain why the oysters changed the chemistry in the jars when the clams did not. Weights are for grouped (n=5) bivalves.
| Totalweight(g) |
Shellweight(g) |
|
| clams |
51.9 |
27.1 |
| oysters |
115.8 |
71.5 |
per oyster wet mass = 8.86 g
Comparison of Dongsen's titrations with ours
| sample |
DXCaCO3mg/L |
DXµmol/kg |
ETSDMµmol/kg |
| low CO2 control1092811 |
198 |
1922 |
2050 |
| low CO2 start exp 092711 |
203 |
1970 |
2045 |
| low CO2 Cg 4-2 092811 |
191 |
1854 |
1769 |
| high CO2 Cg 8-2 092811 |
196 |
1902 |
1884 |
| high CO2 clam 5-2 092811 |
208 |
2019 |
2094 |
| high CO2 start exp 092711 |
205 |
1990 |
2050 |
| CRM 113 |
205 |
1990 |
2224 |
October 3-6, 2011
Secondary Stress Trial
Fed bivalves (see spreadsheet).
September 30, 2011
Secondary Stress Trial
Brought all of our total alkalinity samples up to Friday Harbor to analyze them on the titrator in Moose's lab. Verified accuracy of titrator by running the Dickson standard 3 times - in the beginning, middle, and end of running all of our samples. Oysters caused significant decreases in total alkalinity, regardless of density and feeding status. Clams did not significantly change the alkalinity in their jars. At the end of the experiment, the oysters and clams were put in mesh bags and placed back into the large, round tank with the other oysters and clams. The high CO2 exposed bivalves have zip ties on their bags.
We also dropped 6 samples and 1 CRM off with Dongsen Xue in Forestry to run on his titrator. The samples were : High CO2 Cg 8-2 092811; Low CO2 Cg 4-2 092811; Low CO2 092711 from the start of the experiment; Low CO2 control 1 092811; High CO2 clam 5-2 092811; High CO2 092711 from the start of the experiment. Dongsen reports Total CaCO3 concentrations instead of total alkalinity.