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Troubleshooting DNA/RNA Extraction of Pentagona and Hystera Sea Stars 2

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

Notes

  • Using the Zymo DNA/RNA Miniprep Plus kit
  • Using ceramic beads
  • Using the tissuelyser instead of the vortexer. Manual says different things for DNA and RNA settings so I went in the middle. It says 15 Hz for 30 seconds for DNA, and 2 min at 20-30 Hz for RNA. So I decided to try 1 minute at 15 Hz.
  • Increased incubation time to 2 hours

Sample Preparation

  • Samples:
    • CPBP-006
    • CPDP-003
    • CHSY-002
  • 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 bead tube per sample with 500ul 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
  • CPBP-006 tissue piece and minced piece 1 2
  • CPDP-003 tissue piece and minced piece 3 4
  • CHSY-002 tissue piece and minced piece 5 6
  • Put bead tubes in the tissuelyser for 1 minute at 15Hz
  • Samples were not as broken up as I thought they would be
  • Spun down tubes to get rid of bubbles
  • Added 50ul ProK digestion buffer and 25ul of Proteinase K to each bead tube
  • Vortexed and spun down samples
  • CPBP-006, CPDP-003, and CHSY-002 in order post homogenization and digestion liquid addition: 7
  • Placed all bead tubes in the thermomixer at 55 degrees C shaking 300rpm for 2 hours
  • Tubes looked better after digestion:
  • CPBP-006 8
  • CPDP-003 9
  • CHSY-002 10
  • Removed all the liquid from the bead tubes into new 1.5mL tubes (~450ul)

DNA Extraction

  • Added equal volume (450ul) DNA/RNA lysis buffer to each 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 1 155 RFU - -
Standard 2 16062 RFU - -
CPBP-006 30.2 29.4 29.85
CPDP-003 19.6 19.6 19.6
CHSY-002 21.2 21 21.1
  • RNA
Sample Reading 1 (ng/ul) Reading 2 (ng/ul) Average RNA (ng/ul)
Standard 1 410 RFU - -
Standard 2 8584 RFU - -
CPBP-006 38.6 38.4 38.5
CPDP-003 too low - -
CHSY-002 too low - -

1% Agarose Gel

  • My 3 samples were added in at the end of someone else’s gel due to lots of people wanting to run gels that day
  • Gel was 1% and run at 100V for 1 hour 13

RNA TapeStation

12

Notes

  • The RNA is degraded on the only sample where I got any, I don’t know what happened
Written on October 21, 2020