Updates to HSC DNA Extraction Protocol

Modified protocol formatting from Natalie Ameral’s protocol
Changes include:

  • Materials required section (with # of tubes per sample and suggested tube labels)
  • Purpose added

Purpose

Extracting DNA from horseshoe crab tissue for ddRAD library prep.

Materials required

Tubes needed (per sample)
Tip: Set up and label all tubes before beginning protocol

Tube Suggested label Step used
PBS tube PBS_SampleID Tissue processing
DNA/RNA shield tube DRS_SampleID Tissue processing
Pellet tube SampleID Extraction
Spin column tube 1 (sample loading) SampleID Extraction
Spin column tube 2 (wash) SampleID Extraction
Final DNA tube (1.5ml) SampleID, Date, HSC, DNA, initials Extraction
Strip tubes SampleID Extraction

Cleaning

  • Type 2 DI water squirt bottle
  • 10% bleach squirt bottle
  • 70% ethanol squirt bottle

Tissue processing

  • scalpel handle and 1 blade per sample
  • forceps
  • foil
  • ice bucket
  • sample tubes (thawed in ice bucket)
  • Type 2 DI water (in faclon tube for PBS dilution)
  • PBS
  • DNA/RNA shield
  • proteinase K (in -20 freezer)
  • Blue tissue buffer (Zymo Quick DNA MiniPrep Plus kit)
  • vortexer
  • mini centrifuge
  • thermomixer
  • tubes

Extraction

  • thermomixer
  • centrifuge
  • HCl Tris (100 uL * (# of samples +1)) - warmed in thermomixer
  • tubes
  • spin columns + tubes (Zymo Quick DNA MiniPrep Plus kit
  • liquid waster container
  • Genomic Binding Buffer (Zymo Quick DNA MiniPrep Plus kit)
  • vortexer
  • mini centrifuge
  • DNA pre-wash (Zymo Quick DNA MiniPrep Plus kit)
  • DNA wash buffer (Zymo Quick DNA MiniPrep Plus kit)
  • labeled freezer box (name, date, lab name, DNA extraction, species, project, box #)
  • strip tubes (with sampleID)

Tissue Processing

  1. Take samples out of -80 freezer to thaw slowly in ice bucket
  2. Label 1 1.5mL tube for each sample with “Sample # PBS” and 1 1.5mL tube for each sample with “Sample #”
  3. Add 270ul of DI type II water to each of the PBS tubes
  4. Add 30ul of PBS solution to each of the PBS tubes
    Above 2 steps create 300ul of 1X PBS solution
  5. Add 300ul of DNA/RNA shield to each of the 1.5mL tubes labeled with “Sample #”
  6. Prepare 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”
  7. Place new piece of tinfoil on benchtop and put new scalpel blade on handle (new foil and blade for each sample)
  8. Remove sample from tube
  9. Carefully cut a small piece of tissue away from exoskeleton
  10. Finely chop piece of tissue into thin transparent layer
  11. Place pea sized amount of tissue into tube labeled with corresponding sample number and “PBS”
  12. Place remainder of tissue back into original collection tube
  13. Place remainder of sample back in -80 freezer
  14. Let sample soak for 5-10 minutes while prepping other samples
  15. Remove used scalpel blade from handle and discard in sharps
  16. Repeat steps 6 through 15 for remaining samples
  17. After soaking, move each sample to corresponding tube of DNA/RNA shield
  18. Get proteinase K from -20 freezer and put in ice bucket - currently open tube lives in box with samples that have been processed
  19. Get blue tissue buffer out of Zymo Quick DNA Miniprep Plus kit
  20. Add 150ul of blue tissue buffer to each of the sample tubes
  21. Add 20ul of proteinase K to each sample tube
  22. Vortex and centrifuge each sample on countertop machines for ~10 seconds
  23. Set thermomixer to 55 C
  24. Once warm, put samples in thermomixer for 2 hours at at 1200 rmp

Extraction

  1. While samples are in thermomixer, set up new 1.5mL tubes with “Sample #” (one for each sample)
  2. Turn off thermomixer
  3. Remove samples from thermomixer and centrifuge at 13000rcf for 1 minute
  4. Carefully remove supernatant from above pellet in each tube and deposit liquid only into new tube
  5. Add enough HCL Tris to 1.5ul tube and labeled “Tris HCL” and place in thermomixer on 70 degrees Each sample will need 100ul of Tris HCL so calculate correctly
  6. Set up spin columns for each sample -yellow tubes- in clear collection tubes -no cap- and label with “Sample #”
  7. Get “liquid waste” container
  8. Add 450ul of Genomic Binding Buffer from Zymo kit to each of the tubes containing samples
  9. Vortex and centrifuge each sample for ~5 seconds
  10. Add 700ul of each sample to corresponding spin column set up
  11. Centrifuge tubes at 13000rcf for 1 minute
  12. Pour liquid that collected in bottom tube into liquid waste and place yellow spin column back in corresponding tube
  13. Remove remaining liquid from sample tubes and place in corresponding spin column setup
  14. Centrifuge tubes at 13000rcf for 1 minute
  15. Pour liquid that collected in bottom tube into liquid waste and place yellow spin column back in corresponding tube
  16. Move yellow columns to new collection tubes and discard used ones with tips
  17. Add 400ul DNA pre-wash from Zymo kit to each collection tube setup
    Labelled DNA pre-was with “Pre” on top to avoid confusion
  18. Centrifuge tubes at 13000rcf for 1 minute
  19. Pour liquid that collected in bottom tube into liquid waste and place yellow spin column back in corresponding tube
  20. Add 700ul of DNA Wash Buffer to each spin column
  21. Centrifuge tubes at 13000rcf for 1 minute
  22. Pour liquid that collected in bottom tube into liquid waste and place yellow spin column back in corresponding tube
  23. Add 200ul of DNA Wash Buffer to each spin column
  24. Centrifuge tubes at 13000rcf for 1 minute
  25. Pour liquid that collected in bottom tube into liquid waste and plac yellow spin column back in corresponding tube
  26. Make new 1.5mL tubes labelled with “sample number, Date, horseshoe crab, DNA, NJA”
  27. Move yellow spin columns to labelled tubes created in last step
  28. Discard remaining liquid waste from collection tubes and discard collection tubes in tips
  29. Add 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, place samples in centrifuge with all open caps from 1.5mL tubes facing in same direction and spin at max speed for 1 minute
  31. Remove samples from centrifuge and add 50ul of warmed 10mM Tris HCL to each tube
  32. Let samples incubate for 5 minutes
  33. After incubation, place samples in centrifuge with all open caps from 1.5mL tubes facing in same direction and spin at max speed for 1 minute
  34. Remove samples from centrifuge and discard yellow spin columns in tips
  35. Set up freezer box with name, date, lab name, DNA extraction, and box number or use one that isn’t full
  36. Label new tubes from a set of strip tubes with sample numbers
  37. Remove 8ul from each 1.5mL samples and place in corresponding strip tube
  38. Place samples and strip tubes in Box in -20 upright freezer on Puritz shelf

Maggie’s Qubit Protocol

Written on December 7, 2002