Acropora hyacinthus Eggs and Sperm DNA/RNA Extraction Batch 1

Eggs and sperm from FACE PUF Morea Project

For detailed extraction protocol view post: Protocol for DNA/RNA Extractions of Montipora Coral Larvae Using Zymo-Duet Extraction Kit

Extraction Date: May 24, 2019

Samples

Extraction Number Sample ID Spawning Date Content Sample Quantity (µL) Sample Conserv. Tech
1 796 4/11/2018 Eggs 100 300 uL Shield Erin
2 161 2/11/2018 Sperm 100 300 uL Shield Erin
3 227 2/11/2018 Eggs 100 300 uL Shield Erin
4 203 2/11/2018 Eggs 100 300 uL Shield Erin
5 13 1/11/2018 Sperm 100 300 uL Shield Erin
6 179 2/11/2018 Eggs 100 300 uL Shield Erin
7 191 2/11/2018 Eggs 100 300 uL Shield Erin
8 197 2/11/2018 Sperm 100 300 uL Shield Erin
9 778 4/11/2018 Sperm 100 300 uL Shield Erin
10 209 2/11/2018 Sperm 100 300 uL Shield Erin

Extraction Notes

  • Samples were already preserved in 300µL DNA/RNA Sheild so did not add more
  • Digested for 3 hours and 40 minutes at 55°C 1100 rpm except for samples 13 and 796. These test tubes were too large for the thermomixer and so digested at 55°C speed 35 in Incubator-Genie
  • Added additional 15µL Proteinase K after 3 hours
  • Some lipids left in samples
  • Pelleted at 2300 rcf for 1 min

Qubit

  • Followed Broad Range dsDNA and RNA Qubit protocol
  • All samples were read twice
Extraction # Sample DNA Standard 1 (RFU) DNA Standard 2 (RFU) DNA 1 (ng/µl) DNA 2 (ng/µl) Average DNA RNA Standard 1 (RFU) RNA Standard 2 (RFU) RNA 1 (ng/µl) RNA 2 (ng/ul) Average RNA
1 796 170.9 19464 Too Low Too Low Too Low 383.2 10508 44.6 44.8 44.7
2 161 170.9 19464 46.0 45.8 45.9 383.2 10508 Too Low Too Low Too Low
3 227 170.9 19464 Too Low Too Low Too Low 383.2 10508 179 179 179
4 203 170.9 19464 Too Low Too Low Too Low 383.2 10508 64.2 64.4 64.3
5 13 170.9 19464 30.2 30.0 30.1 383.2 10508 Too Low Too Low Too Low
6 179 170.9 19464 Too Low Too Low Too Low 383.2 10508 26.4 26.4 26.4
7 191 170.9 19464 Too Low Too Low Too Low 383.2 10508 74.0 74.0 74.0
8 197 170.9 19464 5.36 5.34 5.35 383.2 10508 Too Low Too Low Too Low
9 778 170.9 19464 Too Low Too Low Too Low 383.2 10508 Too Low Too Low Too Low
20 209 170.9 19464 82.2 81.8 81.0 383.2 10508 Too Low Too Low Too Low

Tape Station
Quantified on May 28, 2019

TS-Morea-FACE-PUF-Batch-1-Ext-1.png TS-Morea-FACE-PUF-Batch-1-Ext-3.png TS-Morea-FACE-PUF-Batch-1-Ext-4.png TS-Morea-FACE-PUF-Batch-1-Ext-6.png TS-Morea-FACE-PUF-Batch-1-Ext-7.png

Gel

  • Followed PPP Lab Agarose Gel protocol for a small 1.5% agarose gel
  • Used the Thermo Scientific GeneRuler 1 kb Plus DNA Ladder (link)

annotated-Morea-FACE-PUF-gel-batch-1.png

Written on May 24, 2019