Half Volume Montipora Larvae Test DNA/RNA Extraction

Re-Testing Biomin Montipora Larvae and Saving Half the Volume for DNA/RNA Extraction

Goal: Develop a good protocol for extracting high quality DNA and RNA and a good homogenization method for coral larvae/eggs, and save some of the sample to have the possibility to do a second extraction if necessary.
Results: Quantity of DNA and RNA was pretty good for the samples that started with 100 larvae. Quality of DNA and RNA is good. Purity of RNA is not good.
Take-aways: Looks like saving half the initial volume will work for these samples if these yields are enough. We might have to do an extra centrifuge if the wash buffer is carrying over into the elution and causing the poor purity of RNA. Potentially another issue was that I saw there was a noticeable amount of seawater in the tubes before adding the DNA/RNA shield. Unfortunately I didn’t take a picture because I added the shield quickly to avoid thawing.

Sample Info and Protocol Notes

  • Samples are flash frozen larvae in 2mL screw cap tubes. Note, there was some seawater visible in these tubes, probably 100-200ul in there with the larvae
  • Samples are Montipora and from the Biomineralization project
Sample ID Date # Larvae in Original Tube # Larvae in Extraction
399 20180626 50 25
400 20180626 50 25
405 20180626 100 50
406 20180626 100 50
  • After adding the shield and the samples thawed, I spun them down and removed 500ul (1/2 volume) and put in new 1.5mL tubes. I tried to remove half of the larvae with the volume.
  • Followed homogenization method from adult coral DNA/RNA extraction protocol however I proceeded with using the whole volume (~500ul) after that step

Homogenization

  • Took samples one at a time out of -80 and added 1mL of DNA/RNA shield
    399

    400

    405

    406
  • Removed 500ul and ~half the larvae for each tube into new 1.5mL tubes Both tubes 399

    Both tubes 400

    Both tubes 405

    Both tubes 406
  • 1.5mL tubes were placed on ice for the duration of the extraction and then stored at -80C after the extraction finished
  • Added 1/2 of the glass bead tubes to each screw cap sample tube
  • Vortexed for 2 minutes on max speed
  • Samples after vortexing, note they look completely homogenized and there seems to be no layer of lipids:
    399

    400

    405

    406
  • Spun down tubes a few times to lower bubbles
  • Added 50ul Pro K buffer to each tube
  • Added 25ul Proteinase K to each tube
  • Vortexed and spun down samples again
  • Transferred 500ul (most of the volume) to new 1.5mL tubes, avoiding beads. No debris or chunks were visible in the solution

DNA Extraction

  • Warmed 10mM Tris HCl and ultrapure water in thermomixer to 70 degrees C
  • Added equal volume of DNA/RNA lysis buffer to each 1.5mL tube (500ul)
  • Vortexted and spun down tubes
  • Added 700ul of that liquid to a yellow spin column for each sample
  • Centrifuged at 16,000 rcf for 30 seconds
  • Pipetted off the flowthrough into new 5mL tubes for each sample for RNA
  • Repeated last three steps again until all the liquid had gone through the yellow spin columns for each sample
  • Added 400ul prep buffer to each spin column
  • Centrifuged at 16,000 rcf for 30 seconds
  • Poured off flowthrough into kit waste beaker
  • Added 700ul wash buffer to each spin column
  • Centrifuged at 16,000 rcf for 30 seconds
  • Poured off flowthrough into kit waste beaker
  • Added 400ul wash buffer to each spin column
  • Centrifuged for 2 minutes at 16,000 rcf
  • Transferred the spin columns to new labeled 1.5mL tubes
  • Added 50ul warmed 10mM Tris HCl to each column
  • Incubated at room temp for 5 minutes
  • Centrifuged at 16,000 rcf for 30 seconds
  • Again added 50ul warmed 10mM Tris HCl to each column
  • Incubated at room temp for 5 minutes
  • Centrifuged at 16,000 rcf for 30 seconds
  • Saved 10ul in strip tubes for QC, froze the rest in the -20

RNA Extraction

  • Added equal volume fresh 100% ethanol to each of the flow through 5mL tubes from the DNA extraction (1mL)
  • Vortexed and spun down tubes
  • Added 700ul of that liquid to a green spin column for each sample
  • Centrifuged at 16,000 rcf for 30 seconds
  • Poured off flowthrough into kit waste beaker
  • Repeated last three steps again until all of the liquid had gone through the columns
  • Added 400ul wash buffer to each spin column
  • Centrifuged at 16,000 rcf for 30 seconds
  • Poured off flowthrough into kit waste beaker
  • Created the DNase mix:
    • 4 * 75ul DNA digestion buffer = 300ul
    • 4 * 5ul DNase I = 20ul
  • Added 80ul of the DNase mix to each tube and incubated at room temp for 15 minutes
  • Added 700ul wash buffer to each spin column
  • Centrifuged at 16,000 rcf for 30 seconds
  • Poured off flowthrough into kit waste beaker
  • Added 400ul wash buffer to each spin column
  • Centrifuged for 2 minutes at 16,000 rcf
  • Transferred the spin columns to new labeled 1.5mL tubes
  • Added 50ul warmed ultrapure water to each column
  • Incubated at room temp for 5 minutes
  • Centrifuged at 16,000 rcf for 30 seconds
  • Again added 50ul warmed ultrapure water to each column
  • Incubated at room temp for 5 minutes
  • Centrifuged at 16,000 rcf for 30 seconds
  • Saved 7ul in strip tubes for QC, the rest was frozen at -80 (Silver freezer, rack D column 3)

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
399 186 19680 6.3 6.26 6.28
400 - - 5.38 5.3 5.34
405 - - 28.8 28.4 28.6
406 - - 33 32.6 32.8
  • RNA:
Sample Standard 1 Standard 2 RNA 1 ng/ul RNA 2 ng/ul Average ng/ul
399 47.4 819 5.66 5.66 5.66
216 - - 5.68 5.74 5.72
405 - - 32.4 32.4 32.4
406 - - 31.2 31 31.1

NanoDrop

Sample 260/230 260/280
399 0.75 1.78
400 0.77 1.93
405 0.71 2.12
406 1.39 2.06

Full Results:

Traces:

Gel

TapeStation

Written on March 8, 2021