Continuing Through-put Mo'orea Coral Extractions Five

10 DNA only extractions from the first set of Porites and Pocillopora Corals from Mo’orea, 7 to try again, and 3 of Kevin’s Astrangia samples

Using the Zymo Quick-DNA Miniprep Plus kit

Sample Prep

Sample # Type
43 Massive Porites
48 Pocillopora verrucosa
54 Massive Porites
191 Pocillopora verrucosa
164 Pocillopora verrucosa
168 Pocillopora verrucosa
244 Pocillopora verrucosa
269 Massive Porites
275 Massive Porites
280 Massive Porites
210 Massive Porites
52 Massive Porites
272 Massive Porites
306 Massive Porites
285 Massive Porites
58 Massive Porites
39 Massive Porites
H 15 Astrangia poculata
H 21 Astrangia poculata
H 27 Astrangia poculata

Mo’orea new samples

  • Beads were poured into the new sample tubes. The new beads should be easier to pipette the liquid out of them as the do not get sucked up by the p20
  • Samples were homogenized by vortexing for ~60 seconds for all samples
  • Most of the liquid from the tubes was removed by pipetting. This was about 400µl. The tubes contained a small amount of liquid and un-homogenized tissue left, so 200µl of DNA/RNA shield was added to each tube and those were put back into the -20
  • Following recommendations for samples in DNA/RNA Shield from the kit protocol, 200µl of Solid Tissue Buffer and 12µl of Proteinase K were added to each sample
  • Samples were votexed, spun down, and incubated at 55 degrees C for 5 hours shaking at 600rpm

Mo’orea try again samples

  • Whatever tissue was left was in ~300µl and the beads in the -20, another 100µl of DNA/RNA shield was added to make it roughly the same volume as the above samples
  • Tubes were vortexed again for ~30 seconds to homogenize more
  • 200µl of Blue Solid Tissue buffer and 12µl of Proteinase K were added to each sample (beads and all)
  • Samples were votexed, spun down, and incubated at 55 degrees C for 5 hours shaking at 600rpm
  • After digestion, as much liquid was removed as possible, all tubes still had some tissue in them. Sample 39 was very mucusy still
  • Volume was about 500µl

Astrangia samples

  • Samples were already lysed and stored as 500µl in the -80
  • After the Mo’orea samples had digested for 5 hours, these samples were taken out to thaw
  • 500µl of G-DNA binding buffer was added to each tube, vortexed, and spun down
  • Extraction then followed the same way as the Mo’orea samples

Following DNA extraction went along exactly as previous post

Qubit

  • Broad Range dsDNA Qubit protocol
  • All samples were read twice
Sample DNA Standard 1 (RFU) DNA Standard 2 (RFU) DNA 1 (ng/µl) DNA 2 (ng/µl) Average DNA
43 159 17132 46.8 47.4 47.1
48 159 17132 72.4 73.4 72.9
54 159 17132 106 109 107.5
191 159 17132 104 106 105
164 159 17132 72.8 75.2 74
168 159 17132 114 121 117.5
244 159 17132 87.4 88.8 88.1
269 159 17132 24.4 24.6 24.5
275 159 17132 24.8 23.4 24.1
280 159 17132 23.6 21.2 22.4
210 159 17132 10.8 10.9 10.8
52 159 17132 16.7 15.7 16.2
272 159 17132 38.8 38.6 38.7
306 159 17132 12.3 11.8 12
285 159 17132 8.76 9.56 9.16
58 159 17132 10.6 9.56 10.08
39 159 17132 26.4 25.8 26.1
H 15 159 17132 51.4 51.6 51.5
H 21 159 17132 13.1 12.8 13
H 27 159 17132 40 40.4 40.2

Gel Verification

  • A 1.5% agarose gel was ran to check the integrity of the genomic DNA
  • Following the PPP Lab protocol

gel

Written on May 1, 2019