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DNA Only Extraction of 10 Sea Star Samples

DNA Extractions of 10 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:
    • CHSB-004
    • CHSB-006
    • CHSY-004
    • CHSY-010
    • CPAB2-001
    • CPAB2-006
    • CPAB2-007
    • CPDB-005
    • CPDB-006
    • CPAB-007
  • 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
  • Placed tubes in the thermomixer at 55 degrees C for 2 hours shaking at 1200rpm
  • 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 1 188 - -
standard 2 21756 - -
CHSB-004 41.2 40.8 41
CHSB-006 21.2 21 21.1
CHSY-004 40.6 39.8 40.2
CHSY-010 20.6 20.4 20.5
CPAB2-001 34.2 33.8 34
CPAB2-006 31.8 31.6 31.5
CPAB2-007 30 29.8 29.9
CPDB-005 34.2 33.6 33.9
CPDB-006 29.6 29.4 29.5
CPAB-007 34 33.9 33.95

Gel

  • Small 1% Agarose Gel (protocol)
  • Ladder
  • The second row has 2 samples from the previous extraction and 3 samples from Kevin Wong
    gel
Written on December 4, 2020