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

Video and Images


Video of initial experimental setup.

Raw image and video archive

*Zip File* of all timelapse videos and images

Timelapse


22 November 2011

Summary:

-The purpose of Lab 8 was to run qPCR on all of the green urchin samples collected during the experiment.


Materials and Methods:

1. A master mix was made following the chart:

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

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.

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

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


Results:

-Wont have results until after Thanksgiving break.


Conclusion:

-n/a


Next Step:

-analysis data and write research paper.
10 November 2011 (Thursday)
Summary:
-Completed reverse transcription procedures.

Materials and Methods:
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.
Name
 Amount(uL)
M-MLV 5X Reaction Buffer
 5 * 21 = 105ul
dNTPs
 5* 21 = 105ul
M-MLV RT
 21ul
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.

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

Reflection:
-Accidently left unused RNA samples in ice bucket overnight, most likely they are no longer good and will need to be thrown away.
8 November 2011
Summary:
-Completed RNA extraction procedures for 20 Green Urchin Predator Response experiment (GUPR).

Materials and Methods:
-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:
Sample
 ng/ul
 Amount Needed for1ug of RNA
 x2ug per
Reaction
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:
-Wont know the results till we run the gel.
7 November 2011
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
-A copy of my research proposal can be found here.Lab Proposal - Final -Green Urchin Ion Pump.docx
-For the 8th of Nov. lab i will be utilizing techniques from labs 1-4.
20 OCTOBER 2011
Summary:
-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

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
-Lowered tank 3 salinity using DI H2O. Water was slightly warmer then tank water.

-Primer ordered, found complete genome for related species Strongylocentrotus purpuratus.
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:
-Record Experimental data (salinity, number of shell fragments)
-Test RNA extraction on tube feet for ample RNA, if not look to urchin dissection methods.
18 OCTOBER 2011
Summary:
-Experimental set-up complete
-Obtained total 20 green sea urchins, placed in tanks to acclimate.
-Set-up online camera, fully functional.

Materials and Methods:
-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.

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:
-Obtain predators x2, Pisaster ochraceus "Purple sea star"
-Lower salinity
17 OCTOBER 2011
Questions:
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?

Importance:
-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.

Hypothesis:
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.

Experimental Set-up:
-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.

-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.

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

1. Feed Urchins x2 algal pellets ea.
2. Record Data
3. Collect tissue
4. Clean up
 No

Materials:
-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)