RNA and DNA Extractions for Brown/Barott Pdam Project, October 27, 2022

Protocol Link

Extraction of 4 samples from Pocillopora damicornis (Pdam) from Heron Island in RNAlater March 2021 (Kristen Brown, Postdoc in Katie Barott lab)

Samples

Notes

• Today I tested our protocol on these samples. I had to adjust the protocol slightly because they were preserved in RNAlater instead of DNA/RNA shield. I did the following:

  1. Thawed samples on ice.
  2. With sterilized forceps (10% bleach, DEPC water, 70% ethanol, air-dried), I removed one small (5mm x 5mm) fragment from the tube, in which most tubes had 5-6 small fragments of this size. I sterilized forceps between each sample.
  3. I briefly transferred this fragment to a clean 1.5 mL tube on ice to tap off any excess RNAlater and then immediately transferred the fragment to a 1.5 mL screw-cap tube with 800 uL DNA/RNA shield and 0.25 mL of 0.5mm glass beads.
  4. Bead beat for 1-2 minutes on the vortex at max speed. I started off with one minute for all samples but added a minute of bead-beating for samples if the liquid still looked very light-colored after one minute of homogenization.
  5. Briefly spin down and remove 400 uL of supernatant into a clean tube. Spin for 3 mins at 9,000 rcf.
  6. Put original samples and bead tubes back in -80 ºC freezer.
  7. Remove 300 uL of the supernatant into a new tube and continue on with the protocol below (from the Pro K step) as written.

• Other than the adjustments mentioned above, I followed protocol exactly.

• For my records: opened new kit today (Kit labelled 1/2 rec’d 10/4/22), used the columns, collection tubes, and DNA digestion buffer from this new kit.

• I bead-beat RS6D and RF25A for 2 mins instead of 1 min because they were still light-colored after one minute of bead-beating.

Qubit Results

• Used Broad range dsDNA and RNA Qubit Protocol
• All samples read twice, standard only read once

DNA Standards: 183.61 (S1) & 22513.62 (S2)

Sample #	DNA_QBIT_1	DNA_QBIT_2	DNA_QBIT_AVG
RS6D	12.6	12.6	12.6
RS7B	11.8	12	11.9
RF15B	12.7	12.6	12.65
RF25A	6.8	6.68	6.74

RNA Standards: 416.40 (S1) & 9883.91 (S2)

Sample #	RNA_QBIT_1	RNA_QBIT_2	RNA_QBIT_AVG
RS6D	13.2	12.8	13
RS7B	11.2	10.6	10.9
RF15B	15.6	15	15.3
RF25A	nd	nd	0

DNA and RNA Quality Check: Gel, using Kristina’s gel protocol at the bottom of this page.

Next steps

Will use this protocol on the rest of the samples.