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Third Test of Zymo Quick DNA Miniprep Extraction Kit on Horseshoe Crab Tissue

3 More DNA Extraction Tests on Horseshoe Crab Samples from Natalie, Tying Much Smaller Sample Pieces

Samples: 797, 822, and 887 Extraction kit

Sample Prep and Incubation

  • Thawed samples on ice
  • Made 3 1.5mL tubes with 800ul of DNA/RNA Shield
  • 10% bleach, DI and ETOH forceps and scalpel
  • Placed new foil down for each tissue
  • Cut very small pieces of tissue, 1-2cm2, and minced until flat, avoided all cuticle/exoskeleton

797 1 822 2 887 1

  • Added tissue to the 1.5mL tubes with 300ul of DNA/RNA shield, one for each piece
  • Added 150ul of solid tissue buffer to each tube (blue)
  • Added 15ul proteinase K to each tube
  • Vortexed each tube for 10 seconds

  • Spun the tubes down quickly in the minifuge 797 1 822 2 887 1

  • Placed sample tubes in the thermomixer at 55 degrees C shaking at 1200rpm at 9am
  • After 1 hour they looked pretty well digested, let go for 1 more hour though

1

DNA Extraction

  • After incubation, centrifuged tubes at 1300 rcf for 1 min to pellet and insoluble debris
  • Took off supernatant (~450ul) and put into new 1.5mL tubes (pellet small but visible)
  • Added equal volume (450ul) DNA binding buffer to each tube
  • Vortexed and spun down tubes
  • 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 13000 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 13000 rcf for 1 minute and discarded flow through
  • Added 700ul DNA wash buffer to each column
  • Centrifuged 13000 rcf for 1 minute and discarded flow through
  • Added 400ul DNA wash buffer to each column
  • Centrifuged 13000 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 13000 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 1 178 - -
standard 2 19690 - -
797 37.6 37 37.3
822 24.6 24.6 24.6
887 36.4 36 36.2

Gel

gel

Also a TapeStation

TapeStation report showed less “smear” or lighting up around the DNA. Looks pretty good high molecular weight DNA! This extraction protocol works well. I emailed Dr. Heather Bracken-Grissom and her lab (works on regular crabs) soaks the tissue in PBS (phosphate buffered saline) before extraction, a few minutes to a few hours. I might try that.

Written on August 3, 2020