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Troubleshooting DNA/RNA Extraction of Pentagona and Hystera Sea Stars 4

DNA/RNA Extractions on 3 Sea Star Tissue Samples Stored in DNA/RNA Shield, 2 Pentagona and 1 Hystera Homogenizing Using Dremel

Notes

  • Using the Zymo DNA/RNA Miniprep Plus kit
  • Used Dremel with drill bit head for 30 seconds at lowest power setting (5)
  • Incubated for 30 minutes afterwards with proK

Sample Preparation

  • Samples:
    • CPAB1-011
    • CPOI-004
    • CHSY-001
  • Thawed samples on ice bucket
  • Prepared forceps, foil, and razor blades
  • Cleaned forceps with 10% bleach, DI water, 70% ethanol, and RNaseZap before and between each sample
  • Cleaned dremel bit by bathing in beach, DI and ethanol and touching only with forceps between each sample
  • Made 1 1.5mL tube per sample with 300ul of DNA/RNA shield in each
  • Cut a small piece of tissue for each sample then minced with the razor blade until it was flat and could come apart a little bit and placed in tube with shield
  • CPAB1-011 tissue piece and minced piece 1 2
  • CPOI-004 tissue piece and minced piece 3 4
  • CHSY-001 tissue piece and minced piece 5 6
  • CPAB1-011 was homogenized with the dremel first, I went in 10 second increments at 5 speed
  • Before homogenization 7
  • 10 seconds: 7
  • 20 seconds: 8
  • 30 seconds: 9
  • I stopped at 30 seconds because it didn’t look really any more mixed than 20 seconds and I wondered if going longer would mess up the quality of the DNA
  • CPOI-004 before and after 30 seconds of dremel homogenization at 5 speed: 3 4
  • CHSY-001 before and after 30 seconds of dremel homogenization at 5 speed: 5 6
  • Added 30ul prok digestion buffer and 15ul proK
  • Vortexed and spun down tubes
  • Incubated tubes at 55 degrees C for 30 minutes shaking at 500rpm in the thermomixer
  • Tubes after incubation: 6
  • Centrifuged tubes for 2 minutes at maximum speed to pellet debris
  • Pipetted ~320ul of supernatant into new 1.5mL tubes
  • Interestingly, here not all the tissue had pelleted, some was floating, I centrifuged the 2 samples again for 2 min at max speed but it did not help. I have never seen that before: 6

DNA Extraction

  • Added equal volume (300ul) DNA/RNA lysis buffer to each new 1.5mL tube
  • Flicked and inverted tubes to mix and spun down
  • Warmed 10mM Tris HCl pH 8 and nuclease free water in the thermomixer at 70 degrees C
  • Added 700ul of the liquid to yellow spin columns and collection tubes
  • Centrifuged 16,000rcf for 30 seconds
  • Saved the flow through in new 5mL tubes
  • Added the remaining liquid to the yellow spin columns
  • Centrifuged 16,000rcf for 30 seconds
  • Saved the flow through in the 5mL tubes
  • Added 400ul DNA/RNA prep buffer to the yellow spin columns
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded flow through (Zymo kit waste)
  • Added 700ul DNA/RNA wash buffer to the yellow spin columns
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded flow through (Zymo kit waste)
  • Added 400ul DNA/RNA wash buffer to the yellow spin columns
  • Centrifuged 16,000rcf for 2 minutes
  • Transferred spin columns to new 1.5mL tubes for each sample labeled as final DNA tubes
  • Discarded flow through and collection tubes
  • Added 50ul warmed 10mM Tris HCl to the spin columns gently by dripping over the filter
  • Incubated at room temp for 5 minutes
  • Centrifuged 16,000rcf for 30 seconds
  • Added another 50ul warmed 10mM Tris HCl to the spin columns gently by dripping over the filter
  • Incubated at room temp for 5 minutes
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded the spin columns
  • Made strip tubes with 7ul of each sample in them for QC
  • Kept tubes on ice bucket then stored in -20 degree freezer

RNA Extraction

  • Added equal volume (900ul) 100% ethanol to each 5mL tube
  • Vortexed and spun down
  • Added 700ul of the liquid to green spin columns and collection tubes
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded flow through (Zymo kit waste)
  • Repeated addition for the remaining liquid (2 times) to the green spin columns
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded flow through (Zymo kit waste)
  • Added 400ul DNA/RNA wash buffer to the green spin columns
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded flow through (Zymo kit waste)
  • Created the DNase mix:
    • 75ul DNA digestion buffer * 3 = 225ul
    • 5ul DNase I * 3 = 15ul
  • Flicked and spun down mix
  • Added 80ul DNAse mix to each green spin column filter
  • Incubated for 15 minutes at room temp
  • Added 400ul DNA/RNA prep buffer to the green spin columns
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded flow through (Zymo kit waste)
  • Added 700ul DNA/RNA wash buffer to the green spin columns
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded flow through (Zymo kit waste)
  • Added 400ul DNA/RNA wash buffer to the green spin columns
  • Centrifuged 16,000rcf for 2 minutes
  • Transferred spin columns to new 1.5mL tubes for each sample labeled as final RNA tubes
  • Discarded flow through and collection tubes
  • Added 50ul warmed nuclease free water to the spin columns gently by dripping over the filter
  • Incubated at room temp for 5 minutes
  • Centrifuged 16,000rcf for 30 seconds
  • Added another 50ul warmed nuclease free water to the spin columns gently by dripping over the filter
  • Incubated at room temp for 5 minutes
  • Centrifuged 16,000rcf for 30 seconds
  • Discarded the spin columns
  • Made strip tubes with 5ul of each sample in them for QC
  • Kept tubes on ice bucket then stored in -80 degree freezer

QC

Broad Range Qubit for DNA and RNA

Sample Reading 1 (ng/ul) Reading 2 (ng/ul) Average DNA (ng/ul)
Standard 1 189 RFU - -
Standard 2 21594 RFU - -
CPOI-004 19.6 18.9 19.3
CHSY-001 13.3 13.2 13.25
CPAB1-011 18.2 17.9 18.1
  • RNA
Sample Reading 1 (ng/ul) Reading 2 (ng/ul) Average RNA (ng/ul)
Standard 1 403 RFU - -
Standard 2 8723 RFU - -
CPOI-004 17.2 17 17.1
CHSY-001 13.8 13.6 13.7
CPAB1-011 11.8 11.8 11.8

1% Agarose Gel for DNA Quality

  • Followed gel protocol
  • Last three wells in this gel are from this extraction 10

RNA TapeStation

Notes

RNA is not good quality, but also not completely degraded.

Written on November 2, 2020