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Troubleshooting DNA/RNA Extraction of Pentagona and Hystera Sea Stars 5

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

Notes

  • Using the Zymo DNA/RNA Miniprep Plus kit
  • Used Dremel with drill bit head for 20 seconds at lowest power setting (5) on the first two samples
  • Used 1.4mm ceramic beads and the tissuelyser for 2 minutes at 25Hz for the second two samples
  • No incubation, no proteinase K

Sample Preparation

  • Samples:
    • CPAB2-001 dremel
    • CHSY-005 dremel
    • CPAB-006 tissue lyser
    • CPBP-002 tissue lyser
  • 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, RNaseZap and touching only with forceps between each sample
  • Made 1 1.5mL tube per sample with 300ul of DNA/RNA shield for each dremel sample
  • Made 1 ceramic bead tube with 500ul DNA/RNA shield for each tissue lyser sample
  • 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 homogenization tube
  • CPAB2-001 tissue piece and minced piece 1 2
  • CHSY-005 tissue piece and minced piece 3 4
  • CPAB-006 tissue piece and minced piece 5 6
  • CPBP-002 tissue piece and minced piece 5 6
  • Dremel homogenized the first two samples (cleaning bit in between) for 20 seconds on 5 speed
  • Before homogenization with the dremel: 7
  • After homogenization with the dremel: 8
  • Put the bead tubes in the TissueLyser for 2 minutes at 25Hz (balenced with empty bead tubes)
  • After TissueLyser homogenization: 9
  • Spun down the bead tubes in the minifuge to remove bubbles
  • Centrifuged the dremel-homogenized tubes in the centrifuge at max speed for 2 min to pellet debris
  • Still some floating debris in the dremel-tubes after centrifugation, like the previous dremel extraction: 10
  • Avoided all debris and pipetted supernatant from each tube into each a new 1.5mL tube
  • Added equal volume DNA/RNA lysis buffer to each tube (300ul for dremel tubes, 450ul for tissue lyser tubes)

DNA Extraction

  • 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 (600ul and 750ul) 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 1 187 RFU - -
Standard 2 21138 RFU - -
CPAB2-001 13.7 13.5 13.6
CHSY-005 20.8 20.4 20.6
CPAB-006 25.6 25.4 25.5
CPBP-002 32.8 32.6 32.7
  • RNA
Sample Reading 1 (ng/ul) Reading 2 (ng/ul) Average RNA (ng/ul)
Standard 1 398 RFU - -
Standard 2 8374 RFU - -
CPAB2-001 too low - -
CHSY-005 too low - -
CPAB-006 14.6 14.4 14.5
CPBP-002 15.2 14.8 15

1% Agarose Gel for DNA Quality

RNA TapeStation

Notes

RNA quality is really good, just yield is low. DNA quality looks similar between the two methods.

Written on November 5, 2020