Extractions for Moorea Pocillopora spawning 2023

This protocol is based on Maggie’s coral egg/larvae extraction protocol and Kevin’s coral larvae extraction protocol. This post details the info about the extraction steps for larval Pocillopora samples from Moorea 2023. The github for that project is linked here.

Samples

The samples run today were 8, 9, 106, 109, 111, and 112. These samples had ~400 ul of larvae per tube and were preserved in 500 uL of DNA/RNA Shield. They were transported in a freezer box that also included formalin-fixed samples and were stored at 4°C for ~3 months. This photo is post-bead beating:

Materials

• Zymo Quick-DNA/RNA Miniprep Plus Kit HERE Protocol Booklet
• Tris-Ethylenediaminetetraacetic acid (EDTA) 1X buffer for DNA elution
• Heating block capable of heating to 70ºC
• Centrifuge and rotor capable of spinning at 15,000 rcf
• Plastics
  • 3 1.5 mL microcentrifuge tubes per sample
  • 2 PCR tubes per sample
  • 2 Qubit tubes per sample
  • 1 5 mL tube per sample

Protocol

Protocol was followed according to this post. A few differences:

• I added 500 uL of DNA/RNA shield to the samples after thawing them so that the volume in the tubes was ~1 mL of shield. I aliquoted out 500 uL for extractions and saved the remaining fraction in case we need to re-extract.
• In the RNA extraction, I added another wash step with 400 uL of wash buffer after the DNase incubation (i.e., after the incubation, I did 400 uL of prep buffer, 700 uL of wash buffer, 400 uL of wash buffer, and another 400 uL of wash buffer).
• I eluted in 80 uL of either Tris (DNA) or DNA/RNA free water (RNA) instead of 100 uL.
• In the second extraction of the day, I tipped the RNA samples onto the floor when trying to close lids to the caps. This was during the second incubation for RNA elution.

QC

All QC was run the same day as extractions.

Qubit results

Tube.ID	DNA1_ng_uL	DNA2_ng_uL	DNA_avg_ng_uL	RNA1_ng_uL	RNA2_ng_uL	RNA_avg_ng_uL
8	NA	NA	NA	29.6	29.4	29.5
9	NA	NA	NA	46	46.4	46.2
106	3.48	3.46	3.47	11	11	11
109	4.54	4.56	4.55	10.4	10.2	10.3
111	3.96	4	3.98	NA	NA	NA
112	3.52	3.52	3.52	NA	NA	NA

Gel

There are very very very faint bands in the RNA samples for the last 4 samples. I did not get Qubit values for the last 2 samples but hopefully will get tapestation concentrations for them.

Tapestation

See full tapestation report here.

Tapestation looks great! Concentrations are all relatively high, even in the samples that Qubit did not pick up. There are also two bands in the TS gel. These look good to go for library prep.