8 Mo'orea Coral Sample Extractions

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

Using the Zymo Quick-DNA Miniprep Plus kit

Sample Prep

Sample # Site # Date Collected Type
175 5 2018/03/13 Pocillopora verrucosa
177 5 2018/03/13 Pocillopora verrucosa
178 5 2018/03/13 Pocillopora verrucosa
185 5 2018/03/13 Pocillopora verrucosa
197 5 2018/03/13 Massive Porites
200 5 2018/03/13 Massive Porites
205 5 2018/03/13 Massive Porites
303 5 2018/03/13 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 ~30 seconds. Porites samples were homogenized for an extra ~30 seconds
  • Most of the liquid from the tubes was removed by pipetting. This was about 450µl Note that sample 205 did not homogenize noticeably, and did not look like a Porites. The tubes contained a small amount of liquid and un-homogenized tissue left, so those were put back into the -20
  • Following recommendations for samples in DNA/RNA Shield from the kit protocol, 225µl of Solid Tissue Buffer and 15µl 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

DNA Extraction

  1. Centrifuged all tubes at 12,000 rcf for 1 minute to pellet any debris and beads
  2. Removed supernatant into new 1.5mL tubes
  3. Here the proper step is to add 1 volume G-DNA Binding Buffer, but 1 volume (~690µl) G-DNA Wash Buffer was added on accident, this mistake was not noticed until further steps down, but the entire extraction was still processed
  4. 700µl of sample was added to the kit spin column and centrifuged at 12,000 rcf for 1 minute
  5. Collection tubes were discarded
  6. The rest of the coral samples were run through the column in the same way
  7. Added 400µl DNA Pre-Wash Buffer, centrifuged at 12,000 rcf for 1 minute, and discarded the flow through
  8. Added 700µl G-DNA Wash Buffer, centrifuged at 12,000 rcf for 1 minute, and discarded the flow through
  9. Added 200µl G-DNA Wash Buffer, centrifuged at 12,000 rcf for 1 minute, and discarded the collection tube
  10. Columns were transferred to 1.5mL tubes
  11. Added 50µl warmed 70 degrees C 10mM Tris-HCl directly to the column filter and incubated at room temp for 5 minutes
  12. Centrifuged for 1 minute at 12,000 rcf
  13. Repeated steps 11 and 12 one more time

Qubit proceeded immediately to check if the mistake resulted in a failed extraction.

Qubit

  • Broad Range dsDNA Qubit protocol
  • All samples were read twice
  • Standards weren’t recorded in this Qubit because the recorder assumed no DNA would be present
Sample DNA Standard 1 (RFU) DNA Standard 2 (RFU) DNA 1 (ng/µl) DNA 2 (ng/µl) Average DNA
175 - - 4.04 3.96 4
177 - - 5.36 5.3 5.33
178 - - 28.8 28.6 28.7
185 - - 6.58 6.54 6.57
197 - - 44.0 44.6 44.3
200 - - 54.6 54.4 54.5
205 - - too low too low -
303 - - 71.6 71.6 71.6

There is DNA! But, some of the values are very low so I wanted to try extracting from what was left in the tubes with the beads. I also am not sure that the quality is good so I need to run a gel. Samples were stored in 4 degree until the gel could be run the next day.

Gel

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

gel

The quality of the DNA is good actually, but the quantify is low for some samples. Notably 7 didn’t show up, but that is concurrent with the Qubit.

04-11-19 Re-Extraction

  • Added 250µl of DNA/RNA Shield to all of the tubes with the beads and remaining samples
  • Samples were homogenized again for about a minute. Sample 205 still did not break up
  • To make sure ALL the sample got out to maximize DNA yield, I used the sieve method again
  • Sieves/strainers were placed in new labeled 1.5mL tubes
  • About 1/3 of the sample was poured into the sieves and centrifuged briefly as the lid does not fit on the centrifuge
  • The liquid sample went through into the tube but most of the beads stayed in the sieve
  • Repeated pouring the rest of the sample into the sieve and centrifuging quickly
  • 150µl of Solid Tissue Buffer and 10µl 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

DNA Extraction

  1. Centrifuged all tubes at 12,000 rcf for 1 minute to pellet any debris and beads that got through the sieve
  2. Removed supernatant into new 1.5mL tubes
  3. Added 1 volume (460µl) Genomic Binding Buffer to each tube, vortexed and spun down
  4. 700µl, or all for the mussels, of sample was added to the kit spin column and centrifuged at 12,000 rcf for 1 minute
  5. Collection tubes were discarded
  6. The rest of the coral samples were run through the column in the same way
  7. Added 400µl DNA Pre-Wash Buffer, centrifuged at 12,000 rcf for 1 minute, and discarded the flow through
  8. Added 700µl G-DNA Wash Buffer, centrifuged at 12,000 rcf for 1 minute, and discarded the flow through
  9. Added 200µl G-DNA Wash Buffer, centrifuged at 12,000 rcf for 1 minute, and discarded the collection tube
  10. Columns were transferred to 1.5mL tubes
  11. Added 50µl warmed 70 degrees C 10mM Tris-HCl directly to the column filter and incubated at room temp for 5 minutes
  12. Centrifuged for 1 minute at 12,000 rcf
  13. Repeated steps 11 and 12 one more time
  14. Sample tubes were labeled and stored in the 4 degree fridge to quantify the next day

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
175 187 20375 32 32.2 32.1
177 187 20375 36.4 36.6 36.5
178 187 20375 60.6 61.4 61
185 187 20375 38.4 38.4 38.4
197 187 20375 41.4 41.6 41.5
200 187 20375 15.3 15.5 15.4
205 187 20375 too low too low -
303 187 20375 49 49.8 49.4

Still no DNA for 205. When looking at the tissue sample, it does not look like a massive Porites at all, more like algae. It might be a miss-labeled tube.

All tubes from these extraction are labeled A for the first extraction and B for the second and stored in the -20.

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 April 10, 2019