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  2. page home edited This wiki has been developed as a resource for lab personnel and students to access information and…
    This wiki has been developed as a resource for lab personnel and students to access information and publish research activities using an open notebook science based system. The Roberts Lab is in the School of Aquatic and Fishery Sciences within the College of Environment at the University of Washington. More information can be found concerning research, personnel, and outreach on the Roberts Lab Official Webpage.
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Tuesday, November 17

  1. page Becker Lab Notebook edited UWT-Becker Lab10/29/15Finish pK digestionMethods Finishing pK digestion Vials were finger vort…

    UWT-Becker Lab10/29/15Finish pK digestionMethods
    Finishing pK digestion
    Vials were finger vortexed at 9:15am and continued to heat at 56 degrees until the temperature was raised at 11:25am.
    Checking the thermometer and adjusting temperature, the temperature was finally raised to 95 degrees on the thermometer at 11:45am.
    These will heat for an hour at the raised temperature and then cool.
    Took vials off of heat block and set aside to cool briefly.
    Took out multiple other vials to aliquot TC digestions and labeled accordingly (10 vials per TC digestion of Manila clam Pacific geoduck Pacific oyster and Olympia oyster).
    Pipetted out 8 microliters from stock digest of each species and dispensed into respective vial swapping out pipette tips after each aliquot.
    Was going to do the same with the plankton reps and ethanol reps but decided to wait since the digestions were already sitting out for a while. Packed up to take to Seattle tomorrow.
    UWT- Becker LabMcCarthaStart DNA isolation for manila clam spiked plankton samples 10-28-15Created by Michelle McCarthaGoal:Start DNA isolation in prep for qPCR on Friday.Methods:
    Pulled out pK solution Brenda made recently and placed in ice bath for slow melting.
    Checked that all samples left out overnight were completely dried out- which they were- and capped lids.
    Since adding more pk solution may help with any interference with qPCR and may assist in spreading out the data, increased amount of pK solution added to each vial from 700µL to 1000µL.
    Pipetted 1000µL of pK solution into each vial and inverted until sample was suspended in solution.
    Repeated for all samples using new pipette tip for each sample.
    Took samples to heat block which is set at 58C (reading 56C on thermometer) and placed into wells. Placed weight on top to keep lids closed and left for overnight digestion.
    Becker Lab-UWTMcCartha, SmithhislerSpiking plankton and ethanol samples with manila clam larvae for 3 biological reps, as well as preparing template controls of 25 larvae of all four species10/27/15MethodsSpiking plankton and ethanol samples
    Centrifuged 50mL vials for 1 minute at 1300rpm
    I then pipetted 1mL from bottom of the tube and added it to the correspondingly labeled 2mL vial
    I centrifuged the tubes again and then pipetted another 900uL from the bottom and added it to the 2mL vial
    Using new pipette tips every time!
    I visually checked for remaining larvae in the 50mL tubes by adding the contents to a petri dish and rinsing the tube with ethanol.
    I cleaned the petri dish with the 5 step rinse process in between each sample
    Upon making sure there were no remaining larvae in the 50mL tubes, I centrifuged all of the 2mL vials at 2000rpm for 30 seconds.
    I pipetted off as much supernatant as possible without harming the pellet. This was difficult with all of the plankton, so I centrifuged the 2mL vials again and repeated taking off any supernatant.
    Spiking template control samples
    I then spiked TC 2mL vials with 25 larvae
    Vials were labeled accordingly:
    -Cg: 3-6-15 18 days old Pacific Oysters EtOH 50mL vial
    -Pg: 3-6-15 Geoduck EtOH 50mL vial
    -Oly: Hood Canal 160 Oly larvae EtOH 7/16/4
    All 2mL vials were then uncapped and left in the hood to dry overnight.
    UWT-Becker LabSmithhislerTesting different densities of sugar in the sugar gradient method10/24/15, 10/26/15 (counting cont.)Created by: SmithhislerSpiking plankton samples
    I analyzed two, 50mL sample of plankton from Thea Foss (10/08/15) to make sure there were no bivalve larvae.
    I then spiked the samples with 40 Pacific Oyster (18-day old) (03/06/15) larvae using a pipette and rafter slide.
    I centrifuged the 50mL vials for 2 minutes at 1300rpm.
    The supernatant was pipetted off in each vial using a 5000uL pipette in intervals of 5mL at a time and expelled into a petri dish. The tip of the pipette was held by the top and ejected, then the interior and exterior rinsed with 95% ethanol that was added to the petri dish. The supernatant was checked for larvae microscopically.
    Then, for the pellet of each plankton sample (and density), I then swirled the sample up and poured it at an angle gently into the vial with 20mL of syrup so the sample rested on top. I then used the ethanol squirt bottle to rinse as much as possible from the sample to the syrup. This was easiest holding the vial upside down and squirting to the bottom (which is now at the top) and letting the sample flow down and out of the vial.
    I then centrifuged the sample for 2 minutes at 1300rpm.
    Analyzing Sugar Gradient 1.05g/cm3 Density Syrup Trial (syrup prepared 10/09 by BS)
    I began to analyze the sample by pipetting off the phase 1 from the vial and checking for bivalve larvae under the microscope in a counting plate that was rinsed with Micro90 and water and dried with a paper towel. I then rinsed the sample with 95% ethanol into another vial labeled with phase 1,2 and the density of 1.05g/cm3.
    I found 0 larvae in Phase 1.
    I then pipetted out phase 2, checking for larvae. When phase 2 had decreased in volume, I gently added 95% ethanol to the top of the solution to resuspend any remaining phase 2 contents and pipetted them out. However, it was difficult to not pipette up any syrup, and some of the very top of phase 3 was pipetted. I had to use water to then rinse the pipette tip into the counting dish.
    I found 7 larvae in phase 2.
    I then pipetted out only the pellet from the bottom of the tube, leaving phase 3. I attempted to gather all of the materials that had collected at the bottom, but I will check phase 3 in a linear fashion as done previously to make sure I did not miss any larvae when pipetting the pellet.
    I found 33 larvae.
    I then checked phase 3, beginning with the top down, and performing a rinse of the vial with water.
    There were no larvae in phase 3.
    Analyzing Sugar Gradient 1.1g/cm3 Density Syrup Trial (syrup prepared 10/09 by BS)1.1g/cm3
    I began by pipetting off of phase 1 and analyzing for larvae.
    There were no larvae present in phase 1 (the supernatant).
    I then pipetted out phase 2 as much as possible, attempting to avoid grabbing sugar from the top of phase 3. This is very difficult! I added ethanol to the top of the phase 3 surface by running it down the sides of the tube to gather any phase 2 remains. I pipetted out as much as possible, again trying to avoid the sugar, but getting some in the pipette.
    To rinse the pipette tip, I had to use water to get off any sugar residue. This creates an issue with the EtOH and water interaction in the counting dish. However, the current is not too strong that larvae do not settle, so they may still be counted. It is important to realize that the sample from phase 2 is no longer preserved in 95% ethanol because of the rinse with water.
    I found 5 larvae in the phase 2.
    There were many gastropods in this phase.
    I then used a 5mL pipette to gather the pellet and add it to the counting dish. This leaves uncertainty because the pellet is pipetted using visual judgement (as in, there is some pellet debris on the very bottom as well as on the sides of the bottom “V” of the tube”). I attempted to gather as much as possible that I could see, but decided it would be best to pipette out as if I were taking the pellet once more because there was still particles that I could see at/near the bottom (distinguished from the particles ‘floating’ at the top and middle of phase 3). After ejection, the pipette tip for this step has to be rinsed with a generous amount of water. The syrup also changes the view of the polarized lens a little as well. After examining, I added both counts to a vial labeled “pellet”.
    If we ‘miss’ larvae when gathering the pellet, will we want to sort through all of the sugar when performing real samples?
    The first count of the pellet I found 21 larvae.
    The second count of the ‘pellet’, I found 5 larvae.
    I then counted phase 3 in 5mL increments, working from the top down to try to find any remaining linear pattern in larvae distribution throughout the sugar.
    I found only 1 larvae in the last 5mL of phase 3.
    This totals to only 32 larvae (a loss of 8).
    To attempt to understand the loss, I rinsed the sides of the original density vial with water and shook it horizontally. I then added this to a petri dish. There were no larvae.
    Another comment is that I may have mistaken Pacific oly’s for snails.
    Results of Sugar Gradient Method {} The gradient method with a sugarcane syrup density of 1.05g/cm3, after centrifugation for 2 minutes at 1300rpm. {} The gradient method with a sugarcane syrup density of 1.1g/cm3, after centrifugation for 2 minutes at 1300rpm. This photo somewhat shows the pellet distribution in the bottom ‘V’ of the vial. There appears to be particles distributed on the sides of the tube in that location, as well as the flat pellet at the very bottom.
    Future Steps for Sugar Gradient MethodNote: Michelle pointed out that I accidentally used larvae that were previously preserved in DMSO and are now in EtOH. This means the density may be off for the larvae used in these trials. I will attempt a ‘clean’ trial for fast sorting and to see how the larvae move throughout the sugar without interference of plankton.
    Re-try 1.1 density?
    More centrifugation (longer time)? -clearer results, more compact pellet
    Different sized larvae for a mixed sample, maybe try first with just ethanol?
    —> faster sorting
    Less sugar? —>look into papers for information on this
    papers show volumes of 20-25mL

    SAFS- Roberts LabMcCartha
    qPCR Oly primers with Megan's standards 10-23-15Created by Michelle McCartha
    || C || NTC || 25*e1 || 25*e2 || 25*e3 ||
    || D || NTC || 50*e1 || 50*e2 || 50*e3 ||
    NTC || || || |||| || ||
    || F
    NTC || || || |||| || ||
    || G
    TC || || || |||| || ||
    || H
    TC || || || |||| || ||
    Will use 4μL template and 17μL water for all wells except from NTC wells which will have 29μL master mix and 21μL water since it won't have the 4 μL template added to it.
    Centrifuged down plate for 1 min at 2000RPM.
    Standard rep 2 below. Step wise pattern present that corresponds with the standards created. {Screen Shot 2015-10-27 at 12.42.08 PM.png}
    Standard rep three below. Seem to be pretty close here but show stepwise pattern. {Screen Shot 2015-10-27 at 12.42.44 PM.png}
    NTC wells. {Screen{Screen Shot 2015-10-27
    UWS-Roberts LabMcCartha, Smithhisler
    qPCR run on SET 2 geoduck spiked plankton samples10/15/15Created by: Smithhisler
    every well.
    For each well, we began by adding 29uL of MasterMix.
    For NTC, water was then added next at a volume of 21uL.
    Row B template was from the sample was spiked with 5 larvae
    Row C template was from the sample was spiked with 1 larvae
    on the plate. Theplate.The plate was
    at 4C.
    qPCR Cycle Parameters
    Incubate 95C for 2 minutes 30 seconds
    Plate read
    Go to step 2, 39 more times
    began at 11:09am. Brenda11:09am.Brenda filled out
    spiked in sample -Thesesample-These samples contained
    modified pK solution Note:solutionNote: these are
    ran on 10/7/15 -there10/7/15-there were three
    into 6 tubes) -thesetubes)-these samples were
    Results {Screen Shot 2015-10-27 at 1.21.39 PM.png}
    All reps and samples (above). {Screen Shot 2015-10-27 at 1.21.10 PM.png}
    PCR cycleChose PCR reaction for the Pg_4-24-15 program under ‘Bonnie' as listed below:
    Step 1) 95.0°C-10 minutes
    2) 95.0°C-20 seconds StepsecondsStep 3) 65°C-20
    Step 4) 72°C-30 seconds
    Step 5) Repeat steps 2-4 39 more times (40 times total)
    Step 6) 72°C-2 minutes
    at 2:48pm.
    Preparing Gel Electrophoresis
    Michelle prepared a 1.2% agarose solution and poured into gel mold to cool and form.
    Results {} Well order: Ladder, NTC (current primer mastermix), Old primers w/un-dyed larvae DNA (Reps 1, 2, and 3), Current primers with un-dyed larvae DNA (Reps 1,2,3), well 9 is dyed adult DNA and water, then wells 10, 11, and 12 are the reps of dyed larvae DNA and water.
    the old primers. -Theprimers.-The DNA and
    DNA samples.

    StepsThe results
    and Olys.
    UWT- Becker LabMcCarthaStart DNA isolation using modified pK solution 10-14-15Created by Michelle McCarthaGoal:To start cooking spiked plankton samples that were prepared 10-13-15 for overnight digestion.Methods:
    Vials have been sitting in the fumehood for overnight drying since 10-13-15.
    Wells completed for the first plate with no issue.
    replicates. ||
    Used 75 micron and 333 micron mesh to filter sample by first filtering using 75 micron mesh to stop all plankton in range from getting discarded in salt water then filtering what was on 75 micron mesh onto 333 micron mesh using ethanol pouring over a beaker. What was collected in ethanol in the beaker was poured back into the 50ml tube and allowed to settle. Repeated this for all samples.
    Manila clam sequenceUsed google scholar, UW library and NCBI to search for sequences that we can pull primers from. In library search not much was found for primers that were used in the area. Referred back to wight paper and took accession number from there and placed in NCBI. Looked at search results and typed in Venerupis ( RUDITAPES) philippinarum and this came up with numerous results. Countries that these corresponded with include Italy, Korea, China, Quebec, Japan. Took out the (RUDITAPES) part and searched- came up with over 30, 000 results... Continued searching with little success- will continue and engage Steven.
    Not much plankton was collected at all today. Will need to go back out to get plankton that can be used for standard curve testing but the plankton that was collected today will be used isolated to a few tubes and then used to perform density gradient testing.
    Need to keep looking for Manila clam primers to use.
    GoalsFinish counting bivalve larvae in the round 1 pump samples from Port Gamble and Fidalgo Bay.
    I will use this information to determine the maximum larvae in all samples by counting the Grass-Night-Deep samples from all locations.
    "clean" samples.
    I began by rinsing the microscope bench with a 10% bleach solution and wiping dry with paper towels. I then rinsed the bench twice with nanopure water, drying with paper towels.
    13, 17, and 21 would have been replicates of 1, 5 and 9 so were found unnecessary and not made.
    Made an extra 25/10 which was kept in ethanol for later use.
    dry overnight.
    SAFS- Roberts LabMcCartha and Smithhisler
    qPCR on spiked plankton samples round two 9/29/15Created by: McCartha and Smithhisler
    Added water then stock solution. Mixed by pipetting up and down and finger vortexing.
    Finished making master mixes.
    set up
    Second data file saved as 20150918_110407
    No further issues happened aside from the beeping once in a while when the temperature was going up during qPCR.
    Results {qPCR
    Cg Serial
    Image above from qPCR before the machine crashed. {qPCR Cg Serial dilution post shutoff 9-18-15.png}
    Image above from qPCR after the machine crashed and continued to run the curve anyway.
    Image 2 (Below): Gel when changing temperature of annealing stage to 65C. Read from L-R: Row 1 (Top)- Ladder, NTC, TC,1,1,1,2,2,2 Row 2 (Bottom)- Ladder, NTC, TC, 6,6,6,15,15,15. Here you can see that the ladder is working.There is a brighter marker where the template control is which seems to be offset than the other products that are showing up (or it's just too light to see that it's the same length)
    than 1.2%?
    8/7/2015UWT- Becker LabMcCartha
    List of Primers currently in use- with attachment also.
    {} Gel 2 reads from L-R: DNA Ladder, NTC, Rp, Rp, Rp, Cg, Cg, Cg
    Gel 2 shows some discrepancies as all wells show a bit of primer dimer at the farthest point of travel (easily seen as the bands in wells 3 and 4). However, well 2 as the NTC appears to have a specific band of amplification, as well as wells 7 and 8. This brings up questions as to why all of the Cg wells did not have uniform amplification (in this case, well 6 should also have a specific band segment).
    master mix.
    5-25-2015UWT- Becker LabMcCartha
    Finish digestion of all 8 samples for larval DNA and run all samples for PCRCreated by: Michelle McCartha
    Made weigh boat with matching label for each puck.
    Will need to paint pucks prior to weighing them for starting weight before putting them in the field.
    once sieved).
    Using a polarized-lens microscope (3mm across whole FOV @ greatest magnification) searched for bivalve larvae by visually analyzing cross-patterns.
    Found in the first sample 8 bivalve larvae, in the second sample 8 bivalve larvae and in the third sample no larvae. Placed each larvae set in respective 2mL tubes with a little amount of ethanol and set aside for future digestion of tissues for DNA.
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