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Troubleshooting DNA/RNA Extraction of Pentagona and Hystera Sea Stars 3

DNA/RNA Extractions on 3 Sea Star Tissue Samples Stored in DNA/RNA Shield, 2 Pentagona and 1 Hystera Only Incubation

Notes

Sample Preparation

  • Samples:
    • CPBO-003
    • CHSB-011
    • CPDB-008
  • 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
  • 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
  • CPBO-003 tissue piece and minced piece 1 2
  • CHSB-011 tissue piece and minced piece 3 4
  • CPDB-008 tissue piece and minced piece 5 6
  • All tubes pre-incubation: 7
  • Added 30ul ProK digestion buffer and 15ul of Proteinase K to each bead tube
  • Votexed and spun down tubes
  • Placed in thermomixer at 55 degrees C for 3.5 hours shaking at 700rpm
  • All tubes post-incubation 8
  • Used the minifuge to centrifuge and pellet the pieces still in the tube
  • Removed ~300ul of liquid supernatant to new 1.5mL tubes

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 * 4 = 300ul
    • 5ul DNase I * 4 = 20ul
  • 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 1 186 RFU - -
Standard 2 21048 RFU - -
CPBO-003 19.5 19.4 19.45
CHSB-011 11.9 11.8 11.85
CPDB-008 15.8 15.6 15.7
  • RNA
Sample Reading 1 (ng/ul) Reading 2 (ng/ul) Average RNA (ng/ul)
Standard 1 397 RFU - -
Standard 2 8577 RFU - -
CPBO-003 32.8 32.6 32.7
CHSB-011 10.2 10.4 10.3
CPDB-008 25.2 25.6 25.4

1% Agarose Gel for DNA Quality

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

RNA TapeStation

Notes: all RNA is very degraded

Written on October 26, 2020