Montipora Larvae DNA/RNA Extraction 1

Using Larvae Extraction Protocol on 4 of Ariana’s Montipora Coral Larvae Stored in DNA/RNA Shield

Goal: Use optimized extraction method on a few of Ariana’s samples just to be super sure it works
Results: Good DNA and RNA quantity and quality, variable RNA purity
Take aways: This method works, how it was split up for the samples that I saved half worked well. Looks like the timepoint 2 samples might be close to eggs and don’t have enough DNA to extract well

Sample Info:

Sample Timepoint TP Volume Shield in Storage # Larvae
D8 5hpf TP2 1mL 100ul
D14 65hpf TP4 500ul 50ul
D17 93hpf TP5 500ul 50ul
D36 231hpf TP8 300ul 20-30 individuals

Based off of the sample info and how many larvae were present in each tube, save fractions were made for samples D8, D14, and D17, but not for D36

Sample Volume Shield Added at Extraction Save Fraction? Saved Volume
D8 0ul yes 500ul
D14 500ul yes 500ul
D17 500ul yes 500ul
D36 200ul no -

Extraction

  • Extraction protocol followed exactly
  • Samples thawed before extraction:
    D8

    D14

    D17
    D36
  • Samples after additional Shield was added to some samples and some were made into save fractions: D8

    D14

    D17

    D36
  • Extraction proceeded with 500ul of each sample
  • 500ul of DNA/RNA lysis buffer was used for each sample
  • 1mL of 100% ethanol was used for each sample

QC

Qubit

  • Broad Range dsDNA and High Sensitivity RNA Qubit protocol
  • All samples were read twice
  • DNA:
Sample Standard 1 Standard 2 DNA 1 ng/ul DNA 2 ng/ul Average ng/ul
D8 181 19382 too low - -
D14 - - 14.3 14.1 14.2
D17 - - 32 31.6 31.8
D36 - - 17.5 17.3 17.4
  • RNA:
Sample Standard 1 Standard 2 RNA 1 ng/ul RNA 2 ng/ul Average ng/ul
D8 46 815 118 118 118
D14 - - 89 88.6 88.8
D17 - - 50 49.6 49.8
D36 - - 12 11.8 11.9

NanoDrop

Sample 260/230 260/280
D8 1.37 2.10
D14 1.89 2.08
D17 1.70 2.01
D36 0.91 1.68

Full Results:

Traces:

Gel

TapeStation

Sample RIN score
D8 8.1
D14 9.0
D17 8.8
D36 8.8
Written on March 11, 2021