browse by date or tag

Prtocol for Extracting DNA from Horseshoe Crab Tissue

Horseshoe Crab DNA Extraction Protocol

How to use the Zymo Research Quick-DNA Miniprep Plus extraction kit on horseshoe crab leg tissue.

notes
Unless stated: all pipette tips are change between each addition/tube
Don’t let the “spout” of the spin column touch anything, keep resting inside collection tube/1.5mL tube

Sample Preparation and Digestion

  1. Take sample tubes out of the -80 and let thaw (no more than 20 can be extracted at once) on and ice-bead bucket (in the -20 upright freezers)
  2. Write down the sample numbers for the day in your lab notebook
  3. Prepare 2 groups of 1.5mL tubes: 1 for PBS soaking and 1 for sample processing. For the PBS tubes, make one tube per sample and label with the sample number and “PBS.” For the sample processing tubes, make one tube per sample and label with just the sample number. To avoid confusion these tubes can be kept on separate racks
  4. Fill each PBS tube with 90ul of type II DI water and 10ul of 10X PBS solution for a final concentration of 100ul of 1X PBS solution in each tube
  5. Add 300ul of DNA/RNA shield to each sample processing tube and set aside (no need to change tips)
  6. Sterilize forceps and scalpel with 10% bleach, DI water, and 70% ethanol
  7. Lay down new piece of foil and put a new scalpel blade on the scalpel, saving the wrapper for disposal
  8. Use forceps to take out tissue piece and put on foil
  9. Use scalpel blade to cut off “joint” end of the tissue, then make another cross-section to get to the “meat” in the center of the leg. Sometimes the off-white tissue will squeeze out at this step, sometimes you have to cut open the second slice to get at it. Cut a 1-2cm2 piece of all opaque off-white tissue off the piece. Avoid any cuticle. Mince this small piece until it is flat with the scalpel, and place in the PBS tube with the same sample number using the forceps
  10. Let that piece soak for 5-10 minutes (while you cut other tissues), then clean the forceps as in step 6 and transfer the tissue piece into the sample processing tube with the same sample number. Discard the now empty PBS tube in the non-hazardous tip waste bucket
  11. Repeat steps 6-10 for all samples you are doing for the day
  12. Put your samples back in the -80 freezer
  13. Get the proteinase K enzyme from the upright -20 freezer in the “puritz zymo reagents” box and place on the ice bucket
  14. Find the opened Zymo Research Quick-DNA Miniprep Plus kit box and take out the blue solid tissue buffer
  15. Add 150ul of the blue solid tissue buffer to each sample processing tube
  16. Add 15ul of proteinase K enzyme to each sample processing tube
  17. Vortex each tube for 10 seconds at high power
  18. Briefly spin down tubes in the minifuge
  19. Place tubes in the Thermomixer at 55 degrees C, shaking at 1200rpm for 1.5-2 hours
  20. After the incubation is done (most tissue is no longer visible, the liquid is slightly brown, and there are only small flakes of insoluble debris left), place all sample tubes in the tabletop centrifuge and spin at 13,000rcf for 1 minute
  21. After centrifugation, there should be a small pellet of debris stuck to the bottom of the tubes
  22. Make a new set of 1.5mL tubes, one for each sample, with each sample number on the top
  23. Pipette all the supernatant from each centrifuged tube (about 450ul) one at a time into each new sample tube

DNA Extraction

  1. Make a 1.5mL tube full of 10mM Tris HCl and place in the Thermomixer at 70 degrees C
  2. Set up tubes for extractions: Place 1 yellow spin column inside a clear collection tube for each sample. Label the lid of the yellow spin column with the sample numbers
  3. Get the “liquid waste” beaker from the sink by the freezers
  4. Add equal volume (450ul) of Genomic Binding Buffer to each sample tube
  5. Vortex sample tubes for 5 seconds and spin down on the minifuge
  6. Add 700ul of each sample to their labeled yellow spin column
  7. Centrifuge spin columns at 13000 rcf for 1 min (remember to balance centrifuge)
  8. Pour off flow through (liquid now in the collection tube) in the liquid waste beaker
  9. Place spin columns back in their same collection tubes
  10. Add the remaining liquid from each sample to their labeled yellow spin columns
  11. Centrifuge spin columns at 13000 rcf for 1 min
  12. Pour off flow through in the liquid waste beaker
  13. Transfer spin columns to new collection tubes and discard the old ones in non-hazardous tip waste buckets
  14. Add 400ul of DNA Pre-Wash buffer to each spin column
  15. Centrifuge spin columns at 13000 rcf for 1 min
  16. Pour off flow through in the liquid waste beaker
  17. Place spin columns back in their same collection tubes
  18. Add 700ul of g-DNA Wash Buffer to each spin column
  19. Centrifuge spin columns at 13000 rcf for 1 min
  20. Pour off flow through in the liquid waste beaker
  21. Place spin columns back in their same collection tubes
  22. Add 200ul of g-DNA Wash Buffer to each spin column
  23. Centrifuge spin columns at 13000 rcf for 1 min
  24. Write final tubes for each sample: each sample gets a 1.5mL tube with the sample number on the top, and on the side the sample number, “horseshoe crab”, DNA, today’s date, and your initials (plus any other information you want)
  25. Transferr spin columns to opened new final tubes
  26. Pour off last waste and discard collection tube in non-hazardous tip waste bucket
  27. Take warmed 10mM Tris HCl out of the Thermomixer
  28. Add 50ul of warmed 10mM Tris HCl to each spin column by dripping directly over the white filer but not touching the filter tip
  29. Let the tubes incubate with the liquid for 5 minutes
  30. Place the tubes in the centrifuge, place the lids either facing the center or nestled in the empty holes to the right
  31. Centrifuge the tubes at maximum speed for 1 minute
  32. Take tubes out and repeat the previous 5 steps once more for a final volume of 100ul in the tube
  33. Take out spin columns and discard in the non-hazardous tip waste buckets and place tubes on the ice-bead bucket
  34. Set up strip-tubes with the sample number (enough for each sample)
  35. Aliquot 8ul of each sample into their respective 8-strip tube for use in quantity and quality measures
  36. If not qualifying to day, place all tubes in a labeled box in the Puritz shelf in the -20 freezer

Protocols for QC Measures:

Qubit (DNA concentration/yeild)

Agarose Gel Electrophoresis (DNA quality and size)

Written on August 14, 2020