Continuing Through-put Mo'orea Coral Extractions

20 DNA only extractions from Porites and Pocillopora Corals from Mo’orea

Using the Zymo Quick-DNA Miniprep Plus kit

Sample Prep

Sample # Type
40 Massive Porites
41 Pocillopora verrucosa
55 Massive Porites
56 Massive Porites
58 Massive Porites
59 Pocillopora verrucosa
65 Pocillopora verrucosa
67 Massive Porites
75 Massive Porites
159 Pocillopora verrucosa
167 Pocillopora verrucosa
171 Pocillopora verrucosa
208 Massive Porites
220 Massive Porites
222 Massive Porites
232 Pocillopora verrucosa
241 Pocillopora verrucosa
273 Massive Porites
304 Massive Porites
312 Massive Porites
  • New Beads were poured into 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

Following DNA extraction went along exactly as previous post

Qubit

  • Broad Range dsDNA Qubit protocol
  • Qubit standards and master mix working stock was made by Kevin Wong
  • All samples were read twice
Sample DNA Standard 1 (RFU) DNA Standard 2 (RFU) DNA 1 (ng/µl) DNA 2 (ng/µl) Average DNA
40 209 23359 26.6 26.4 26.5
41 209 23359 88.8 88.2 88.5
55 209 23359 29.8 29.6 29.7
56 209 23359 21.6 21.4 21.5
58 209 23359 8.42 8.34 8.38
59 209 23359 30.4 30.2 30.3
65 209 23359 56.4 56 56.2
67 209 23359 4.9 4.8 4.85
75 209 23359 35 34.8 34.9
159 209 23359 54.2 53.8 54
167 209 23359 64.2 63.8 64
171 209 23359 60 60 60
208 209 23359 14.8 14.6 14.7
220 209 23359 15.8 15.6 15.7
222 209 23359 41 40.6 40.8
232 209 23359 74.2 73.8 74
241 209 23359 44 43.8 43.9
273 209 23359 9.22 9.14 9.8
304 209 23359 54.2 53.8 54
312 209 23359 29.4 29.2 29.5

67 and 220 were re-dos, so there might not have been that much left in the first place. I need to do a gel to see how the quality is.

Gel Verification

  • A 1.5% agarose gel was ran to check the integrity of the genomic DNA
  • Following the PPP Lab protocol
  • Samples 67 and 220 were not run on the gel

gel

Written on April 18, 2019