Horseshoe Crab DNA Extraction - Version 2

Horseshoe Crab DNA Extraction with Zymo Quick DNA Miniprep Plus kit

Date Performed: September 12th, 2020
Tissue samples processed: 447, 448, 449, 450, 452, 453, 454, 455

Processed samples following steps from previous Extraction Post with a few minor changes:
*Skipped PBS soaking step *Increased Proteinase K from 15 - 20ul *Used less tissue

Tissue Processing

  1. Took 3 samples out of -80 freezer to thaw slowly in ice bucket
  2. Labeled 3 1.5mL tubes with “Sample #”
  3. Added 90ul of DI type II water to each of the 3 PBS tubes
  4. Added 300ul of DNA/RNA shield to each of the 1.5mL tubes labeled with “Sample #”
  5. Prepared scalpel handle and forceps by cleaning with 10% bleach solution, DI II water, and 70% ethanol solution From this step forward this cleaning process will simply be referred to as “cleaning protocol”
  6. Placed new piece of tinfoil on benchtop and put new scalpel blade on handle
  7. Removed first sample from tube
  8. Carefully cut a small piece of tissue away from exoskeleton
  9. Finely chopped piece of tissue into thin transparent layer
  10. Placed half pea sized amount of tissue into tube labeled with corresponding sample number
  11. Placed remainder of tissue back into original collection tube
  12. Placed remainder of sample back in -80 freezer
  13. Removed used scalpel blade from handle
  14. Repeated steps 6 through 15 for remaining samples
  15. Got proteinase K from -20 freezer and put in ice bucket
  16. Got blue tissue buffer out of Zymo Quick DNA Miniprep Plus kit
  17. Added 150ul of blue tissue buffer to each of the sample tubes
  18. Added 20ul of proteinase K to each sample tube
  19. Vortexed and centrifuged each sample on countertop machines for ~10 seconds
  20. Set thermomixer to 55 C
  21. Once warm, put samples in thermomixer at 2:30 PM at 1200 rmp

Extraction

  1. While samples are in thermomixer, set up 3 new 1.5mL tubes with “Sample #”
  2. Samples incubated for 1.5 hours
  3. Removed samples from thermomixer and centrifuged at 13000rcf for 1 minute
  4. Carefully removed supernatant from above pellet in each tube and deposited liquid only into new tube
  5. Added 400ul of HCL Tris to 1.5ul tube and labeled “Tris HCL” and placed in thermomixer on 70 degrees Each sample will need 100ul of Tris HCL so calculate correctly
  6. Set up 3 spin columns -yellow tubes- in clear collection tubes -no cap- and labelled with “Sample #”
  7. Got “liquid waste” container
  8. Added 450ul of Genomic Binding Buffer from Zymo kit to each of the tubes containing samples
  9. Vortexed and centrifuged each sample for ~5 seconds
  10. Added 700ul of each sample to corresponding spin column set up
  11. Centrifuged tubes at 13000rcf for 1 minute
  12. Poured liquid that collected in bottom tube into liquid waste and placed yellow spin column back in corresponding tube
  13. Removed remaining liquid from sample tubes and placed in corresponding spin column setup
  14. Centrifuged tubes at 13000rcf for 1 minute
  15. Poured liquid that collected in bottom tube into liquid waste and placed yellow spin column back in corresponding tube
  16. Moved yellow columns to new collection tubes and discarded used ones with tips
  17. Added 400ul DNA pre-wash from Zymo kit to each collection tube setup Labelled DNA pre-was with “Pre” on top to avoid confusion
  18. Centrifuged tubes at 13000rcf for 1 minute
  19. Poured liquid that collected in bottom tube into liquid waste and placed yellow spin column back in corresponding tube
  20. Added 700ul of DNA Wash Buffer to each spin column
  21. Centrifuged tubes at 13000rcf for 1 minute
  22. Poured liquid that collected in bottom tube into liquid waste and placed yellow spin column back in corresponding tube
  23. Added 200ul of DNA Wash Buffer to each spin column
  24. Centrifuged tubes at 13000rcf for 1 minute
  25. Poured liquid that collected in bottom tube into liquid waste and placed yellow spin column back in corresponding tube
  26. Made 3 new 1.5mL tubes labelled with “sample number, Date, horseshoe crab, DNA, NJA”
  27. Moved yellow spin columns to labelled tubes created in last step
  28. Discarded remaining liquid waste from collection tubes and discarded collection tubes in tips
  29. Added 50ul of warmed 10mM Tris HCL to each tube to incubate for 5 minutes Carefully drip liquid directly onto white disc in yellow tube
  30. After incubation, placed samples in centrifuge with all open caps from 1.5mL tubes facing in same direction and spun at max speed for 1 minute
  31. Removed samples from centrifuge and added 50ul of warmed 10mM Tris HCL to each tube
  32. Let samples incubate for 5 minutes
  33. After incubation, placed samples in centrifuge with all open caps from 1.5mL tubes facing in same direction and spun at max speed for 1 minute
  34. Removed samples from centrifuge and discarded yellow spin columns in tips
  35. Set up freezer box with name, date, lab name, DNA extraction, and box number 1
  36. Labelled 3 new tubes from a set of strip tubes with sample numbers
  37. Removed 8ul from each 1.5mL samples and placed in corresponding strip tube
  38. Placed samples and strip tubes in Box 1 in -20 upright freezer on Puritz shelf
Written on September 12, 2020