Symbiont Integration Metabolomics Processing (20210321 - 20210414)
Project: Symbiont Integration
Goal
To prepare lab materials and process metabolomic extractions on multiple life stages of M. capitata samples for the Symbiont Integration experiment using the larval protocol.
Laboratory preparation
- Autoclaved all glass ware.
- Wiped down all counter tops and fumehood with 10% bleach solution.
- Obtained dry ice.
- Took aliquots of master extraction buffer and 15% ammonium bicarbonate solutions for each day.
- Make a 10% alkaline soap solution in a 1000mL beaker for dounces
- Prep the centrifuge so it is at 4°C
- Labelled all temporary tubes (2 per sample for Flash Rozen samples, 3 per sample for plug samples) and loading vials (2 per sample)
Protocol
Flash Frozen Samples
- Put glass dounce on dry ice
- Grab sample from -80°C freezer
- Remove any residual sea water and immediately add 500uL of the extraction buffer to the tube
- Pipette mix and transfer to glass dounce
- Add an additional 500uL to the sample tube and add to glass dounce
- Incubate on dry ice for 5 minutes
- Homogenize with glass pestle for ~1 minute
- If the sample is visibly pigmented and opaque, add an additional 500uL of extraction buffer to rinse the glass pestle into the dounce.
- If not visibly pigmented, skip this step
- Strain the homogenate through the 100um cell strainer into the 50mL falcon tube
- Transfer the strained homogenate to a new and labelled 1.5 mL tube
- Centrifuge for 10 minutes at 15000 rcf at 4°C
- Transfer 500uL of the supernatant to a new, labelled 1.5 mL tube
- Add 44uL of cold 10% ammonium bicarbonate
- Vortex and spin down
- Load 100uL into each labelled loading vial (duplicates)
- Store all tubes (temporary and loading vials) in a labelled box in the -80°C freezer.
Juveniles on Plugs Samples
- Sterilize a razor blade for each sample with 10% bleach, DI water, then 70% ethanol.
- Add 500uL of the extraction buffer to a labelled 1.5 mL tube and keep on dry ice.
- Grab the plug from the freezer and quickly chip off/scrape off the juvenile into the cold 1.5mL centrifuge tube
- If the scraped tissue is stuck on the edge of tube, use a 200p pipette tip to push the tissue into the extraction buffer.
- pipette mix and transfer to glass dounce
- Add an additional 500uL to the sample tube and add to glass dounce
- Incubate on dry ice for 5 minutes
- Homogenize with glass pestle for ~1 minute
- If the sample is visibly pigmented and opaque, add an additional 500uL of extraction buffer to rinse the glass pestle into the dounce.
- If not visibly pigmented, skip this step
- Strain the homogenate through the 100um cell strainer into the 50mL falcon tube
- Transfer the strained homogenate to a new and labelled 1.5 mL tube
- Centrifuge for 10 minutes at 15000 rcf at 4°C
- Transfer 500uL of the supernatant to a new, labelled 1.5 mL tube
- Add 44uL of cold 10% ammonium bicarbonate
- Vortex and spin down
- Load 100uL into each labelled loading vial (duplicates)
- Store all tubes (temporary and loading vials) in a labelled box in the -80°C freezer.
Sample Processing dates
20210321
Samples:
| Original Tube Label | Life Stage | Extraction Number | New Vial Label |
|---|---|---|---|
| F23 | Larvae 4 | 1 | #1-A-20210321 |
| F25 | Larvae 5 | 2 | #2-A-20210321 |
| F33 | Recruit 1 | 3 | #3-A-20210321 |
| F2 | Egg Fertilized | 4 | #4-A-20210321 |
| F12 | Larvae 1 | 5 | #5-A-20210321 |
| F9 | Larvae 1 | 6 | #6-A-20210321 |
| F6 | Embryo 1 | 7 | #7-A-20210321 |
| F1 | Egg Fertilized | 8 | #8-A-20210321 |
| F20 | Larvae 3 | 9 | #9-A-20210321 |
| F35 | Recruit 1 | 10 | #10-A-20210321 |
Notes:
- All samples were extracted with 1500uL of extraction buffer.
All samples were loaded into duplicate vials (A and B) and additional extracts were saved in the same box and stored at -80°C.
Box Label: Symbiont integration Metabolomic Extractions 20210321 - KW/AH/Putnam Box 1
Freezer: New grey/blue -80°C
20210325
Samples:
| Original Tube Label | Life Stage | Extraction Number | Extraction Buffer Volume (uL) | New Vial Label |
|---|---|---|---|---|
| F16 | Larvae 2 | 11 | 1500 | #11-A-20210325 |
| F4 | Egg Fert | 12 | 1500 | #12-A-20210325 |
| F28 | Larvae 5 | 13 | 1500 | #13-A-20210325 |
| F32 | Larvae 6 | 14 | 1000 | #14-A-20210325 |
| F27 | Larvae 5 | 15 | 1000 | #15-A-20210325 |
| F31 | Larvae 6 | 16 | 1000 | #16-A-20210325 |
| F19 | Larvae 3 | 17 | 1500 | #17-A-20210325 |
| F3 | Egg Fert | 18 | 1500 | #18-A-20210325 |
| F10 | Larvae 1 | 19 | 1500 | #19-A-20210325 |
Notes:
- I noticed some samples had low pigment coloration (Larvae 5/6/recruits) possibly due to low sample input. Therefore, I reduced the amount of extraction buffer to potentially increase the signal intensity.
All samples were loaded into duplicate vials (A and B) and additional extracts were saved in the same box and stored at -80°C.
Box Label: Symbiont integration Metabolomic Extractions 20210321 - KW/AH/Putnam Box 1
Freezer: New grey/blue -80°C
20210407
Samples:
| Original Tube Label | Life Stage | Extraction Number | Extraction Buffer Volume (uL) | New Vial Label |
|---|---|---|---|---|
| PLUG | Recruit 1 | 20 | 1500 | #20-A-20210407 |
| F39 | Recruit 2 | 21 | 1500 | #21-A-20210407 |
| F26 | Larvae 5 | 22 | 1000 | #22-A-20210407 |
| F7 | Embryo 1 | 23 | 1500 | #23-A-20210407 |
| F24 | Larvae 4 | 24 | 1500 | #24-A-20210407 |
| F40 | Recruit 2 | 25 | 1000 | #25-A-20210407 |
| F18 | Larvae 3 | 26 | 1500 | #26-A-20210407 |
| F38 | Recruit 2 | 27 | 1000 | #27-A-20210407 |
| F36 | Recruit 1 | 28 | 1000 | #28-A-20210407 |
| F8 | Embryo 1 | 29 | 1500 | #29-A-20210407 |
Notes:
All samples were loaded into duplicate vials (A and B) and additional extracts were saved in the same box and stored at -80°C.
Box Label: Symbiont integration Metabolomic Extractions 20210321 - KW/AH/Putnam Box 2
Freezer: New grey/blue -80°C
20210408
Samples:
| Original Tube Label | Life Stage | Extraction Number | Extraction Buffer Volume (uL) | New Vial Label |
|---|---|---|---|---|
| PLUG | Recruit 3 | 30 | 1500 | #30-A-20210408 |
| PLUG | Recruit 2 | 31 | 1000 | #31-A-20210408 |
| PLUG | Recruit 1 | 32 | 1000 | #32-A-20210408 |
| PLUG | Recruit 3 | 33 | 1000 | #33-A-20210408 |
| PLUG | Recruit 2 | 34 | 1000 | #34-A-20210408 |
| PLUG | Recruit 1 | 35 | 1000 | #35-A-20210408 |
| PLUG | Recruit 3 | 36 | 1000 | #36-A-20210408 |
| PLUG | Recruit 1 | 37 | 1000 | #37-A-20210408 |
| PLUG | Recruit 3 | 38 | 1000 | #38-A-20210408 |
| F5 | Embryo 1 | 39 | 1500 | #39-A-20210408 |
Notes:
- Made new extraction buffer
- #31 had very little tissue. Will need to do another recruit 2 replicate just in case.
All samples were loaded into duplicate vials (A and B) and additional extracts were saved in the same box and stored at -80°C.
Box Label: Symbiont integration Metabolomic Extractions 20210321 - KW/AH/Putnam Box 2
Freezer: New grey/blue -80°C
20210414
Samples:
| Original Tube Label | Life Stage | Extraction Number | Extraction Buffer Volume (uL) | New Vial Label |
|---|---|---|---|---|
| PLUG | Recruit 2 | 40 | 1000 | #40-A-20210414 |
| PLUG | Recruit 2 | 41 | 1000 | #41-A-20210414 |
| PLUG | Recruit 2 | 42 | 1000 | #42-A-20210414 |
| F17 | Larvae 3 | 43 | 1500 | #43-A-20210414 |
| F11 | Larvae 1 | 44 | 1500 | #44-A-20210414 |
| F37 | Recruit 2 | 45 | 1000 | #45-A-20210414 |
| F15 | Larvae 2 | 46 | 1500 | #46-A-20210414 |
| F34 | Recruit 1 | 47 | 1000 | #47-A-20210414 |
Notes:
- #40 was one of the plugs that was in a broke whirl pack, but re-identified as a 72 hps plug. This is also the plug I split with Maggie for her RNA extraction.
- #42 was one of the plugs that was in a broke whirl pack, but re-identified as a 72 hps plug. This sample is in place of extraction #31.
All samples were loaded into duplicate vials (A and B) and additional extracts were saved in the same box and stored at -80°C.
Box Label: Symbiont integration Metabolomic Extractions 20210321 - KW/AH/Putnam Box 3
Freezer: New grey/blue -80°C
Symbiont pellet pictures on the haemocytometer
These pictures are from the pellet after the 10 minute centrifugation. I used sample #4 from this extraction set to photograph.
