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Troubleshooting DNA/RNA Extraction of Pentagona and Hystera Sea Stars 6

DNA/RNA Extractions on 3 Sea Star Tissue Samples Stored in DNA/RNA Shield, 2 Pentagona and 1 Hystera, Using the Tissuelyser for 1 Minute at 15Hz and Incubating at Room Temperature for 15 Minutes

Notes

  • Using the Zymo DNA/RNA Miniprep Plus kit
  • Used 1.4mm ceramic beads and the tissuelyser for 1 minute at 15Hz, stopped at 30 seconds and moved the tubes to the other side of the sample holder in the lyser.
  • Incubated at room temp with the proteinase K for 15 minutes shaking at 550rpm

Sample Preparation

  • Samples:
    • CPBP-008
    • CPAB-001
    • CHSB-004
  • 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 ceramic bead tube with 800ul DNA/RNA shield for each tissuelyser sample (as per the updated kit recomendations)
  • 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 their respective bead homogenization tube
  • CPBP-008 tissue piece and minced piece 1 2
  • CPAB-001 tissue piece and minced piece 3 4
  • CHSB-004 tissue piece and minced piece 5 6
  • Bead tubes before tissuelyser homogenization: 7
  • Put bead tubes in the tissuelyser for 30 seconds at 15Hz
  • Orientation of bead tubes in holders: 8
  • After 30 seconds, changed orientation of bead tubes in holders: 9 10
  • Ran the tissuelyser again for 30 seconds at 15Hz
  • Spun down the bead tubes in the minifuge to remove bubbles
  • After homogenization: 11
  • Added 80ul proteinase K digestion buffer and 40ul proteinase K to each bead tube
  • Briefly vortexed and spun down sample tubes
  • Placed in the thermomixer for 10 minutes shaking at 550rpm at 22 degrees C temp (room temp)
  • At 10 minutes checked on the status of the tissue: 12
  • Saw still some tissue so decided to go to 15 minutes
  • Status of tissue at 15 minutes: 13
  • Tissue looked pretty broken down, hard to tell if it was just in between the beads
  • Removed all the supernatant (~830ul) into new 1.5mL tubes
  • Centrifuged tubes for 1 minute at 16,00rcf
  • Noticed pellet after centrifugation: 14
  • Removed liquid (~830ul) into new 5mL tubes
  • Added equal volume (830ul) DNA/RNA lysis buffer to the 5mL tubes
  • Vortexed and spun down tubes

DNA Extraction

  • Flicked and inverted tubes to mix and spun down
  • Warmed 10mM Tris HCl pH 8 in the thermomixer at 70 degrees C note did not warm H2O this time
  • 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
  • Repeated addition, centrifugation, and saving of flowthrough for the remaining amount of liquid (3 total spins)
  • 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 (1660ul) 100% ethanol to the 5mL tubes of flowthrough
  • 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, centrifugation, and saving of flowthrough for the remaining amount of liquid (5 total spins)
  • 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 room temp 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 room temp 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 1 199 RFU - -
Standard 2 21978 RFU - -
CPBP-008 28.4 28 28.2
CPAB-001 26.6 26.4 26.5
CHSB-004 13.3 13.1 13.2
  • RNA
Sample Reading 1 (ng/ul) Reading 2 (ng/ul) Average RNA (ng/ul)
Standard 1 404 RFU - -
Standard 2 8406 RFU - -
CPBP-008 too low - -
CPAB-001 11 10.8 10.9
CHSB-004 too low - -

1% Agarose Gel for DNA Quality

RNA TapeStation

Written on November 12, 2020