--- title: "Spawn night protocol" subtitle: "For embryonic development of Montipora capitata rice corals" author: "Sarah Tanja" date: '03/26/2024' --- # Prep \*\*Filtering leachate & dilution took about 45 minutes\*\* ## Make Stock Leachate First make 400mL of each stock solution from the 1000 mg/L prepared leachate - [ ] Make 10 mg/L stock: 1000 mg/L \* V1 = 10 mg/L \* 400mL V1 = 4mL 400mL of 10mg/L stock = 4mL of 1000mg/L stock + 396mL of FSW - [ ] Make 1mg/L stock: 10mg/L \* V1 = 1mg/L \* 400mL V1 = 40mL 400mL of 1mg/L stock = 40mL of 10mg/L stock + 360mL of FSW - [ ] Make 0.1 mg/L stock: 1mg/L \* V1 = 0.1mg/L \* 400mL V1 = 40mL 400mL of 0.1mg/L stock = 40mL of 1mg/L stock + 360mL of FSW 2. Then dilute the stock to each treatment vial/jar For a 20mL scintillation vial for coral embryo experiments with a final target volume of 15mL: **1mg/L HIGH treatment** 10mg/L \* V1 = 1mg/L \* 19mL V1 = 1.9mL 19mL of 1mg/L leachate = 17.1mL of FSW + 1.9mL of 10mg/L stock **0.1 mg/L MID treatment** 1mg/L \* V1 = 0.1mg/L \* 20mL V1 = 12mL 20mL of 0.1mg/L leachate = 18mL of FSW + 2mL of 1mg/L stock **0.01 mg/L LOW treatment** 0.1mg/L \* V1 = 0.01mg/L \* 18mL V1 = 2mL 20mL of 0.01mg/L leachate = 18mL of FSW + 2mL of 0.1mg/L stock ## ZFIX Going from 18.5% to 4% in a \~100mL final volume! We want a total of 100 mL volume 0.185 \* V1 = 0.04 \* 100mL V1 = 21.62mL To get 100mL of 4% ZFIX: Add 21.62 mL of 18.5% ZFIX to 78.38mL of (0.22micron) filtered sea water ::: callout-important Jill Ashey says it's important to use FSW to dilute your ZFIX! This is for long-term storage (up to 6 months) of embryos in anywhere from 4-20% ZFIX or paraformaldehyde (PFA) ... the FSW may keep your embryos from lysing so that you can preserve their entegrity for microscopy. ::: ## Freeze (& shield) To preserve embryos for RNA extraction and sequencing we will first submerge them in Zymo DNA/RNA Shield, and then pronptly transfer them to a -80C freezer. Here, we won't be dunking them in LN2. The combined DNA/RNA Shield and the quick freeze (due to small size) they will experience in the -80 are together sufficient for preserving RNA. We will first very gently use a tip-clipped transfer pipette to transfer embryos to our 0.5mL screw-cap cryo-tubes. Wewill then carefully decant any excess FSW out of the tube, and then add 500uL of Zymo RNA/DNA Shield. Gently invert cryo-tubes a few times to ensure embryos are fully submerged. Once cryo-tubes are filled with their samples, arrange them in wax paper boxes and promptly shift them to -80°C freezer for storage until samples are ready to be processed. # Spawn Night ### 19:00 - 19:30 - [ ] Move each colony to a numbered/named cambro chamber bin or 5 gallon bucket - [ ] Ensure each colony has enough headspace so that bundles can rise to the surface - [ ] Reduce blue holding tank water level to below the lip of the chambers - [ ] Decant water in chambers such that bouyant bundles don't spill out ### 19:30 - 20:00 - [ ] Organize the following collection materials at the spawning tubs: - [ ] 50mL falcon tubes (1 for ea. colony) filled with 30mL of 0.22micron FSW - [ ] Tip-clipped transfer pipettes - [ ] Red Headlamps (new batteries) - [ ] Printed waterproof spawn collection data sheet - [ ] Organize the following collection materials at the dry lab bench: - [ ] 20mL scintillation vials prepped with treatments - [ ] 50mL falcon tube rack (for working with active bundle-bundle crosses) - [ ] Tip-clipped transfer pipettes (for moving bundles to scintillation vials) - [ ] Printed waterproof bundle-bundle cross metadata sheet (for recording parent colonies of crosses) ### 20:50 - 21:20 - [ ] Observe for setting (polyp expansion and bundle visibility) via red headlamp - [ ] Find a spawner! - [ ] Collect 24 bundles from each spawning colony and transfer them to a pre-labeled 50mL falcon tube with 30mL of FSW - [ ] Once we have at least 2 colonies worth of bundles, 1 person will move with any filled falcon tubes to the dry lab bench and begin bundle-bundle crosses - [ ] 1 person will remain and continue to collect bundles from colonies, periodically transferring them to the bundle-bundle crossing station at the dry lab bench ### 21:20 - 22:00 - [x] Cross fertilize by taking one bundle each from two distinct colonies and transferring them to the same scintillation vial - [x] Record the parent colonies of each cross on the embryonic development metadata sheet - [x] Only transfer intact bundles! Once eggs break up and begin to hydrate, the spawn party is over ### 22:00 - 22:30 - [x] Observe scintillation vials for bundle breakup and egg hydration - [ ] Record the times when the first and last vials experience hydration - [x] First Egg Hydration Time\_\_\_\_\_22:00\_\_\_\_\_\_\_\_ - [ ] Last Egg Hydration Time\_\_\_\_\_22:\_\_\_\_\_\_\_\_\_\_ - [ ] Average the times above , and add 4 hours, 9 hours, and 14 hours - [ ] Average Egg Hydration Time\_\_\_22:20\_\_\_\_ - [ ] 4 hours post-fertilization[^1]\_\_\_\_\_02:20\_\_\_\_\_ - [ ] 9 hours post-fertilization\_\_\_\_\_\_07:20\_\_\_\_\_ - [ ] 14 hours post fertilization\_\_\_\_\_\_12:20\_\_\_ - Set alarms for \~40 minutes prior to the times above [^1]: - [ ] (& on July 6th make another batch of leachate!) ### 22:30 - 23:00 - [ ] Return each colony to blue tank and remove chambers - [ ] Return water height to normal - [ ] Clean up anything left outside ### 23:00 - 01:45 - [ ] 2.5 hr nap time! ### 02:15 - 03:30 - [ ] ZFIX 4hpf samples (4Z) - [ ] Use a transfer pipette to gently move embryos from scintillation vials to microcentrifuge tubes - [ ] Decant any excess FSW with transfer pipette - [ ] Add \~1mL of 4% ZFIX to each microcentrifuge tube in the fume hood! - [ ] Gently invert each capped scintillation vial to mix the ZFIX into the filtered seawater and sample - [ ] Record time of fixin' : \_\_\_\_\_\_\_\_\_\_ - [ ] Add 8-12 hours to fixin' time to transfer from ZFIX to 70% ethanol: \_\_\_\_\_\_\_\_\_\_\_\_ - [ ] Set fixed vials in 4C fridge - [ ] Freeze 4hpf samples (4F) - [ ] Gently transfer live embryos to cryo-vials - [ ] Decant excess FSW using a transfer pipette - [ ] Add 500uL of Zymo DNA/RNA Shield - [ ] Promptly transfer to -80C freezer - [ ] Make PVC Leachate (on July 6th!) ### 03:30 - 06:45 - [ ] 3.25 hr nap time! ### 07:15 - 08:30 - [ ] ZFIX 9hpf samples (9Z) - [ ] Use a transfer pipette to gently move embryos from scintillation vials to microcentrifuge tubes - [ ] Decant any excess FSW with transfer pipette - [ ] Add \~1mL of 4% ZFIX to each microcentrifuge tube in the fume hood! - [ ] Gently invert each capped scintillation vial to mix the ZFIX into the filtered seawater and sample - [ ] Record time of fixin' : \_\_\_\_\_\_\_\_\_\_ - [ ] Add 8-12 hours to fixin' time to transfer from ZFIX to 70% ethanol: \_\_\_\_\_\_\_\_\_\_\_\_ - [ ] Set fixed vials in 4C fridge - [ ] Freeze 9hpf samples (9F) - [ ] Gently transfer live embryos to cryo-vials - [ ] Decant excess FSW using a transfer pipette - [ ] Add 500uL of Zymo DNA/RNA Shield - [ ] Promptly transfer to -80C freezer ### 08:30 - 11:45 - [ ] 3.25 hr nap time! ### 12:15 - 13:30 - [ ] ZFIX 14hpf samples (14Z) - [ ] Use a transfer pipette to gently move embryos from scintillation vials to microcentrifuge tubes - [ ] Decant any excess FSW with transfer pipette - [ ] Add \~1mL of 4% ZFIX to each microcentrifuge tube in the fume hood! - [ ] Gently invert each capped scintillation vial to mix the ZFIX into the filtered seawater and sample - [ ] Set fixed vials in 4C fridge - [ ] Freeze 14hpf samples (14F) - [ ] Gently transfer live embryos to cryo-vials - [ ] Decant excess FSW using a transfer pipette - [ ] Add 500uL of Zymo DNA/RNA Shield - [ ] Promptly transfer to -80C freezer ::: callout-tip You can do this! :::