Acropora Eggs and Sperm DNA/RNA Extraction Batch 2

Eggs and sperm from FACE PUF Morea Project 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 28, 2019

Samples

Extraction Number Sample ID Spawning Date Content Sample Quantity (µL) Sample Conserv. Tech
11 784 4/11/2018 Eggs 100 300 uL Shield Erin and Emma
12 215 2/11/2018 Eggs 100 300 uL Shield Erin and Emma
13 760 4/11/2018 Eggs 100 300 uL Shield Erin and Emma
14 790 4/11/2018 Sperm 100 300 uL Shield Erin and Emma
15 19 1/11/2018 Eggs 100 300 uL Shield Erin and Emma
16 185 2/11/2018 Sperm 100 300 uL Shield Erin and Emma
17 173 2/11/2018 Sperm 100 300 uL Shield Erin and Emma
18 754 4/11/2018 Sperm 100 300 uL Shield Erin and Emma
19 167 2/11/2018 Eggs 100 300 uL Shield Erin and Emma
20 221 2/11/2018 Sperm 100 300 uL Shield Erin and Emma

Extraction Notes

  • Altough samples were already preserved in 300µL DNA/RNA Sheild, I added an additional 300µL of shield to prevent RNA/DNA degradation during digest
  • Digested for 4 hours 55°C 1100 rpm except for samples 784, 19, 754, 167 and 760. 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 2 hours
  • After 4 hours the samples had barely digested. To lyse the cells we micropestled each egg sample with sterilized micropestles and microcentrifuge tubes
  • To accomodate the use of extra shield, we added 750µL Lysis buffer instead of 345µL (Step 3 DNA Extraction). Additionally, we mixed 1000µL EtOH instead of 700µL in 5µL tubes (Step 1 RNA Extraction) and repeated steps 6-7 once.

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
11 784 193.8 20337 Too Low Too Low Too Low 392.6 10735 96.6 96.6 96.6
12 215 193.8 20337 Too Low Too Low Too Low 392.6 10735 101 101 101
13 760 193.8 20337 Too Low Too Low Too Low 392.6 10735 109 109 109
14 790 193.8 20337 58.6 58.0 58.3 392.6 10735 10.2 10.2 10.2
15 19 193.8 20337 Too Low Too Low Too Low 392.6 10735 153 153 153
16 185 193.8 20337 76.0 75.8 75.9 392.6 10735 Too Low Too Low Too Low
17 173 193.8 20337 87.8 87.6 87.7 392.6 10735 Too Low Too Low Too Low
18 754 193.8 20337 53.2 53.2 53.2 392.6 10735 11.0 11.0 11.0
19 167 193.8 20337 Too Low Too Low Too Low 392.6 10735 121 121 121
20 221 193.8 20337 132 132 132 392.6 10735 12.0 12.2 12.1

Tape Station
Tape stationed on May 29, 2019 by Emma Strand

TS-Morea-FACE-PUF-Batch-2-Ext-11.png TS-Morea-FACE-PUF-Batch-2-Ext-12.png TS-Morea-FACE-PUF-Batch-2-Ext-13.png TS-Morea-FACE-PUF-Batch-2-Ext-14.png TS-Morea-FACE-PUF-Batch-2-Ext-15.png TS-Morea-FACE-PUF-Batch-2-Ext-16.png TS-Morea-FACE-PUF-Batch-2-Ext-17.png TS-Morea-FACE-PUF-Batch-2-Ext-18.png TS-Morea-FACE-PUF-Batch-2-Ext-19.png

Gel

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

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

Written on May 28, 2019