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DNA Only Extraction of 3 Sea Star Samples

DNA Extractions of 3 Hystera and Pentagona With the Zymo Research Quick-DNA Miniprep Plus Kit

Notes: followed kit recommendations for samples stored in DNA/RNA Shield and used and intermediate incubation time of 2 hours

Sample Preparation and Digestion

  • Samples:
    • CPOI-007
    • CPBP-002
    • CHSY-006
  • Thawed samples on ice bucket
  • Prepared forceps, foil, and razor blades
  • Cleaned with 10% bleach, DI water, and 70% ethanol before and between each sample
  • Prepared a 1.5mL tube for each sample with 300ul 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 their respective 1.5mL tube
  • Added 150ul of the blue solid tissue buffer to each sample processing tube
  • Added 10ul of proteinase K enzyme to each sample processing tube
  • Vortexed and spun down tubes
  • Samples before incubation:
  • CPOI-007 1
  • CPBP-002: 1
  • CHSY-006 1
  • Placed tubes in the thermomixer at 55 degrees C for 2 hours shaking at 1200rpm
  • Samples after incubation for 2 hours: 1
  • CPBP-002: 1
  • CHSY-006 1
  • Placed all sample tubes in the tabletop centrifuge and spun at 16,000rcf for 1 minute
  • Removed ~450ul of supernatant into new 1.5mL tubes
  • Added 450ul of Genomic DNA Binding Buffer to each new tube
  • Vortexed and spun down tubes

DNA Extraction

  • Set up 1 spin column and collection tube per sample
  • Warmed 10mM Tris HCl to 70 degrees C in the thermomixer
  • Added 700ul of each sample to their labeled spin columns
  • Centrifuged at 16000 rcf for 1 min
  • Discarded flow through
  • Added the rest of the liquid to the spin columns and centrifuged in the same way
  • Transferred columns to new collection tubes
  • Added 400ul DNA pre-wash buffer to each column
  • Centrifuged 16000 rcf for 1 minute and discarded flow through
  • Added 700ul g-DNA wash buffer to each column
  • Centrifuged 16000 rcf for 1 minute and discarded flow through
  • Added 200ul g-DNA wash buffer to each column
  • Centrifuged 16000 rcf for 1 minute and discarded flow through
  • Made 1.5mL tubes labeled completely with sample names and information
  • Transferred spin columns to those 1.5mL tubes
  • Added 50ul of warmed 10mM tris HCl by dripping to each spin column filter
  • Incubated columns for 5 minutes
  • Centrifuged columns for 1 min at 16000 rcf
  • Repeated last 3 steps once
  • Placed tubes on ice afterwards

Qubit

  • Broad range dsDNA qubit (protocol)
  • Amounts are in ng/ul and samples were read twice
Sample Reading 1 Reading 2 Average DNA (ng/ul)
standard 1 183 - -
standard 2 19507 - -
CPOI-007 33.6 33.2 33.4
CPBP-002 33 33.4 33.2
CHSY-006 40.4 40.6 40.5

Gel

gel

Written on November 20, 2020