Green+Sea+Urchin+Predator+Response

-Derek Brady -Miranda Kyle -Igor last name
 * Group Members: Notebook Links**


 * Video and Images**

[|Video of initial experimental setup.]

Raw image and video archive

[|*Zip File* of all timelapse videos and images]

Timelapse media type="youtube" key="lfb2rIbkuWc?version=3" height="360" width="480"

__ **22 November 2011** __ -The purpose of Lab 8 was to run qPCR on all of the green urchin samples collected during the experiment. 1. A master mix was made following the chart: 2. Total of 20 samples; x2 negative controls = 22 total x2 for duplicates = 44 + 2 for error = 46 samples 3. Placed x6 (8-well) well strips with labels 1-6 and my initials. 4. Master-mix was vortex-ed for 45 secs to ensure mixing. 5. 23ul of master mix was placed into each of the 46 wells. 6. The below chart corresponds to each cDNA sample in its respective well. -2uL of cDNA was added to each respective well. -2uL of DI H2O was added to Negative Controls. 7. Caps were placed on all x6 well strips. 8. qPCR conditions: 1. 95°C for 10 minutes 2. 95°C for 15s 3. 55 °C for 15 s 4. 72°C for 30 s (+ plate read) 5. Return to step 2 39 more times 6. 95°C for 10s -Wont have results until after Thanksgiving break. -n/a -analysis data and write research paper.
 * Summary: **
 * Materials and Methods: **
 * ~ Component || Volume(uL) || Multiplier || TotalVolume(uL) ||
 * ~ x2 Immomix || 12.5 || x46 || 575 ||
 * ~ Syto-13 dye (50uM) || 1 || x46 || 46 ||
 * ~ Up Stream Primer || 1.25 || x46 || 57.5 ||
 * ~ Down Stream Primer || 1.25 || x46 || 57.5 ||
 * ~ Di H2O || 7 || x46 || 322 ||
 * ~ Strip Number ||~ Well#1 ||~ 2 ||~ 3 ||~ 4 ||~ 5 ||~ 6 ||~ 7 ||~ 8 ||
 * ~ 1 || Neg Control || Neg Control || C1 || C1 || C2 || C2 || C3 || C3 ||
 * ~ 2 || C4 || C4 || C5 || C5 || C6 || C6 || P1 || P1 ||
 * ~ 3 || P2 || P2 || P3 || P3 || P4 || P4 || P5 || P5 ||
 * ~ 4 || P6 || P6 || P7 || P7 || S1 || S1 || S2 || S2 ||
 * ~ 5 || S3 || S3 || S4 || S4 || S5 || S5 || S6 || S6 ||
 * ~ 6 || S7 || S7 || Neg Control || blank || blank || blank || blank || blank ||
 * Results: **
 * Conclusion: **
 * Next Step: **

__ **10 November 2011 (Thursday)** __ -Completed reverse transcription procedures.
 * Summary: **

Reverse Transcription- Reverse Transcription Procedures: 1. Added 2ug of TOTAL RNA to appropriate PCR tube. 2. Added 1ul of Oglio DT, no H2O 3. Incubated for 5min and prepared master mix. 4. Added 11ul to each tube and placed in thermocycler. x1- 90C for 5 min,x40 cycles of (90C for 15s, 55C for 15s, 72C for 15s), x1- 71 for 5 mins.
 * Materials and Methods: **
 * Name || Amount(uL) ||
 * M-MLV 5X Reaction Buffer || 5 * 21 = 105ul ||
 * dNTPs || 5* 21 = 105ul ||
 * M-MLV RT || 21ul ||

-Will run gel on Tuesday 15th November.
 * Conclusion: **

-Accidently left unused RNA samples in ice bucket overnight, most likely they are no longer good and will need to be thrown away.
 * Reflection: **

__ **8 November 2011** __ -Completed RNA extraction procedures for 20 Green Urchin Predator Response experiment (GUPR).
 * Summary: **

-RNA extraction part 1: 1. Labeling is as follows (C1-6) = Controls, (P1-7) = Predator Only, (S1-7) = Salinity Dec. and Predator. 2. Added 500ul Tri-reagent and homogenized. 3. Once homogenized we added 500ul of additional Tri-reagent, then vortexed for 15secs. (We did not turn on the heating block as it was not required to assist in separation.) 4. Incubated for 5-6 mins and then added 200ul Chloroform under the fume hood. 5. Vortex for 30s. 6. Incubated for 15s and then placed into 4C centrifuge at maximum speed for 15min. (8 of 20 samples did not completely separate and had to be reran through the vortex and then centrifuge again.) 7. Transferred the aqueous phase to new 1.5ml tube. Disposed rest of material in waste jar. (Do all this under fume hood!) 8. Added 500ul isopropanol. to each sample and inverted several times. 9. Incubated for 10 mins then spun down at max speed for 8 mins. 10. Removed supernatant. 11. Added 1ml of 75% ETOH and spun at 7500 rpm for 5 mins. 12. Removed EtOH and dried inside tubes with chemwipe, did not touch RNA pellet. 13. Suspended the pellet in 100uL of 0.1%DEPC-H2O. 14. Ran Nano-Drop on all 20 samples. Results of Nano-Drop: Reaction || -Wont know the results till we run the gel.
 * Materials and Methods: **
 * < Sample ||< ng/ul ||< Amount Needed for1ug of RNA ||< x2ug per
 * < C1 ||< 40.6 ||< 24.6 ||< 49.2 ||
 * < C2 ||< 31.2 ||< 32.1 ||< 64.2 ||
 * < C3 ||< 9.3 ||< 107.5 ||< 215 ||
 * < C4 ||< 22.5 ||< 44.4 ||< 88.8 ||
 * < C5 ||< 17.0 ||< 58.8 ||< 117.6 ||
 * < C6 ||< 22.5 ||< 44.4 ||< 88.8 ||
 * < P1 ||< 61.5 ||< 16.3 ||< 32.6 ||
 * < P2 ||< 17 ||< 58.8 ||< 117.6 ||
 * < P3 ||< 28.2 ||< 35.5 ||< 71 ||
 * < P4 ||< 53.2 ||< x ||< 37.6 ||
 * < P5 ||< 21.3 ||< x ||< 93.9 ||
 * < P6 ||< 28.1 ||< x ||< 71.2 ||
 * < P7 ||< 47.7 ||< x ||< 41.9 ||
 * < S1 ||< 27.6 ||< x ||< 72.5 ||
 * < S2 ||< 60.9 ||< x ||< 32.8 ||
 * < S3 ||< 25.8 ||< x ||< 77.5 ||
 * < S4 ||< 55.4 ||< x ||< 36.1 ||
 * < S5 ||< 47.0 ||< x ||< 42.6 ||
 * < S6 ||< 47.2 ||< x ||< 42.4 ||
 * < S7 ||< 35.5 ||< x ||< 56.3 ||
 * Conclusion: **

__**7 November 2011**__ -A copy of my research proposal can be found here. -For the 8th of Nov. lab i will be utilizing techniques from labs 1-4.
 * Timeline for Derek's experiment into urchin ion cotransporter (Na-K-2Cl):**
 * Date ||  Experimental Plan  ||
 * Exp Day 1 – Wednesday, October 19, 2011 || Experimental set-up, organism collection, primer design, begin acclimatization of specimens. Feed 2-3 algal pellets each urchin. ||
 * Exp Day 2 – Thursday, October 20, 2011 || Begin Experiment 1330. Add predators to respective tanks 2 & 3. Lower tank 3 salinity using DI water to 20psu. Feed 2-3 algal pellets each urchin. ||
 * Exp Day 3 – Friday, October 21, 2011 || Monitor shell fragments collected, Feed 2-3 algal pellets each urchin. ||
 * Exp Day 4 – Saturday, October 22, 2011 || Monitor shell fragments collected, change water, check salinity. Feed 2-3 algal pellets each urchin. ||
 * Exp Day 5 – Sunday, October 23, 2011 || Monitor shell fragments collected, Feed 2-3 algal pellets each urchin. ||
 * Exp Day 6 – Monday, October 24, 2011 || Monitor shell fragments collected, Feed 2-3 algal pellets each urchin. Water change, check salinity. ||
 * Exp Day 7 – Tuesday, October 25, 2011 || Collect tissue samples and store in -80C freezer. ||
 * Lab Week 1 – Tuesday November 1 – November 7 || Begin RNA extraction and Isolation of tube feet tissue. ||
 * Lab Week 2 – Nov 8th – Nov 14th || Run PCR procedures ||
 * Lab Week 3 – Nov 15th – Nov 21st || Run qPCR procedures ||
 * Lab Week 4 – Nov 22nd – Nov 30th || Review data/ write paper/ design presentation ||

__**20 OCTOBER 2011**__ -Lowered Salinity of tank 3. -Added x2 Pisaster ochraceus to 2nd and 3rd tanks. -Added clams to 2nd and third tanks for sea star food. -Angled 1st tank for better view. -Added barrier between 1st and 2nd tanks. -Ordered Primer
 * Summary:**

-Lowered tank 3 salinity using DI H2O. Water was slightly warmer then tank water.
 * Methods:**
 * Tank || Temp (C) || Salinity ||
 * 1-Control || 11.6 || 30.5 ||
 * 2-Predator || 11.5 || 31.2 ||
 * 3-Predator and Low Salinity || 11.5 || 21.8 ||

-Primer ordered, found complete genome for related species Strongylocentrotus purpuratus. -Record Experimental data (salinity, number of shell fragments) -Test RNA extraction on tube feet for ample RNA, if not look to urchin dissection methods.
 * Name || Gene || Seq || Designed By || Bps || GC% || Tm || Species || Acension # || Location ||
 * Sp_NaK2Cl_F || Na-K-2Cl cotransporter 1 (NKCC1) || TGAGATACGACACGCCACCA || Derek B. || 20 || 55 || 55.43 || Strongylocentrotus purpuratus || NM_001113236.1 || NCBI: GenBank ||
 * Sp_NaK2Cl_R || Na-K-2Cl cotransporter 1 (NKCC1) || GTTCTCTTTCGGGGCAGCTT || Derek B. || 20 || 55 || 54.79 || Strongylocentrotus purpuratus || NM_001113236.1 || NCBI: GenBank ||
 * Next Step:**

__**18 OCTOBER 2011**__ -Experimental set-up complete -Obtained total 20 green sea urchins, placed in tanks to acclimate. -Set-up online camera, fully functional.
 * Summary:**

-x3 tanks -x3 air stones and pumps -Switched third tank stressor from OA to low salinity -placed sand to 1in depth all tanks. -placed shell fragments in each tank, to one side.
 * Materials and Methods:**

-Obtain predators x2, __Pisaster ochraceus__ "Purple sea star" -Lower salinity
 * Acclimatization Step:**
 * Tank || Temperature (C) || Salinity (ppt) || Number of Urchins ||
 * 1-Control || 11.5 || 30.8 || x6 ||
 * 2-Predator || 11.6 || 31.5 || x7 ||
 * 3-Low Salinity/Predator || 11.8 || 29.5 || x7 ||
 * Next Step:**

__**17 OCTOBER 2011**__ 1. Does exposure to predators result in the covering response observed in green sea urchin (Strongylocentrotus droebachiensis)? 2. Does exposure to light result in the covering response observed in green sea urchins? 3. What mechanism activates the covering response?
 * Questions:**

-It is important to understand how organisms react to predatory conditions, especially of the sea urchin. This organism when left unchecked and without predators has the ability to desolate habitats such as kelp forests which in turn impacts dozens if not hundreds of other species.
 * Importance:**

1. Exposure to predators will increase the covering behavior in time of reaction, duration, and number of shell fragments utilized. 2. Exposure to light will result in the covering behavior. 3. The mechanism is a behavior that has multiple activators such as light and predator proximity.
 * Hypothesis:**

-Total of 3 tanks will be used with 7 sea urchins within each tank (N=21, n=7). All tanks will be recorded either visually or with video equipment to observe the covering behavior duration and time of response. All tanks will contain the same weight, type, and consistency of shell/ rock fragments, consistency refers to the average size of the shell fragments. Roughly 1 inch of sand will be placed in the bottom of each tank.
 * Experimental Set-up:**

-Within all tanks we will be recording time, duration, and number of shell fragments collected by the specimens.

-The question if light activates the covering behavior will be answered by placing a red light within the experimental set-up and observing at night as the lights will be turned off and on to simulate day and night cycles.

-The first tank will act as control containing only seawater and 7 urchins for the duration of the experiment. The first tank will also need to be sealed or isolated so that no possible water from the experimental tanks crossover as predator excrement within the water may alter results. -The second tank will contain 7 urchins and will be exposed to a predator, by transferring water from the predators tank to the tank with the urchins. This should provide enough effluents and chemicals from the predator to illicit a response in the green sea urchins. -The third tank will contain 7 urchins that will be exposed to a predator (same as the second tank) but will also contain a lower salinity content (15ppt) to observe the synergistic effects of predator response and decreasing salinity on urchin covering behavior.

-The experiment is planned to for 120 hours from Thursday beginning at 1330 to Tuesday ending at 1330.

2. Add predator water (x)ml 3. Lower Salinity of tank 3 to (15) ppt || No || 2. Record Data || No || 2. Record Data 3. Change out water || Yes || 2. Record Data || No || 2. Record Data 3. Change out water || Yes || 2. Record Data 3. Collect tissue 4. Clean up || No ||
 * Time Schedule:**
 * Date || Time || Time of Feeding || Tasks || Water Change ||
 * 20OCT2011 Thursday || 1330-1630 || 1400 - Derek, Igor || 1. Feed Urchins x2 algal pellets ea.
 * 21OCT2011 Friday ||  || 1330 - Derek || 1. Feed Urchins x2 algal pellets ea.
 * 22OCT2011 Saturday ||  || 0900-1100 || 1. Feed Urchins x2 algal pellets ea.
 * 23OCT2011 Sunday ||  || 0900-1100 || 1. Feed Urchins x2 algal pellets ea.
 * 24OCT2011 Monday ||  || 1330 - || 1. Feed Urchins x2 algal pellets ea.
 * 25OCT2011 Tuesday ||  ||   || 1. Feed Urchins x2 algal pellets ea.

-x3 fish tanks -x3 oxygen hoses -x3 oxygen pumps -x3 oxygen diffusers -x21 green sea urchins -x1 predator (Sea star) -x1 Salinity Monitor -x 1 yd black paper -x1 camera -Sand -Shell Fragments -Food (Algal Pellets)
 * Materials:**