Acropora eggs/sperm DNA and RNA Extractions

E. Strand, E. Chille 20190528 DNA/RNA Extractions, Qubit
E. Strand 20190529 Tape Station
E. Strand, M. Schedl 20190529 Ship Samples

20190528 DNA / RNA Extractions, Qubit

DNA and RNA was extracted from 10 Acropora eggs and sperm samples from the Moorea FACE PUF project using Zymo-Duet Extraction Kit and following the attached protocol: DNA and RNA Extractions

Original Sample List:

Extraction # Sample_ID Spawn_Date* Coral_ID Sample_Content Sample_Quantity Sample_conserv Label Storage_location
11 784 4/11/2018 TBR7_52 Eggs 100 uL 300 uL shield E52-S Rhode Island
12 215 2/11/2018 TBR7_55 Eggs 100 uL 300 uL shield E55-S Rhode Island
13 760 4/11/2018 FR3_61 Eggs 100 uL 300 uL shield E61-S Rhode Island
14 790 4/11/2018 TBR7_50 Sperm 100 uL 300 uL shield S50-S Rhode Island
15 19 1/11/2018 FR3_62 Eggs 100 uL 300 uL shield E62-S Rhode Island
16 185 2/11/2018 FR3_64 Sperm 100 uL 300 uL shield S64-S Rhode Island
17 173 2/11/2018 FR3_63 Sperm 100 uL 300 uL shield S63-S Rhode Island
18 754 4/11/2018 FR3_61 Sperm 100 uL 300 uL shield S61-S Rhode Island
19 167 2/11/2018 TBR7_53 Eggs 100 uL 300 uL shield E53-S Rhode Island
20 221 2/11/2018 FR3_60 Sperm 100 uL 300 uL shield S60-S Rhode Island
  • Spawn date is likely written in Canadian/European format with the day/month/year. Double check with K. Wong.

Extraction notes:

  • Samples entered digest at 11:30 am.
  • Added additional 300 uL of RNA/DNA shield to prevent RNA/DNA degradation in digest.
  • Added additional 15 uL of Proteinase K after 2 hours.
  • 5 samples were in the incubator genie because the tubes were too large for the thermomixer.
  • Samples in the incubator genie looked more digested than the thermomixer so we moved all of the samples into the incubator genie at 3:30 pm.
  • We used a micropestle to break down the eggs around 3:45-4:00 pm.
  • We used 5 mL tubes to mix RNA and ethanol and spun down the entire sample (there was a larger amount of liquid because of the extra RNA/DNA shield).

DNAse I:
Buffer = 75 x 10 = 750 uL
DNAse = 5 x 10 = 50 uL

Qubit:
Buffer = 199 x 12.2 = 2427.8 uL
Reagent = 12.2 uL

Qubit Results:
Protocol: Qubit

Sample_ID Coral ID BR DNA 1 BR DNA 2 DNA Average BR RNA 1 BR RNA 2 RNA Average
Standard 1 NA 193.8 NA NA 392.6 NA NA
Standard 2 NA 20337 NA NA 10735 NA NA
11 784 too low too low too low 96.6 96.6 96.6
12 215 too low too low too low 101 101 101
13 760 too low too low too low 109 109 109
14 790 58.6 58 58.3 10.2 10.2 10.2
15 19 too low too low too low 153 153 153
16 185 76 75.8 75.9 too low too low too low
17 173 87.8 87.6 87.7 too low too low too low
18 754 53.2 53.2 53.2 11 11 11
19 167 too low too low too low 121 121 121
20 221 132 132 132 12 12.2 12.1
Written on May 28, 2019