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Testing Zymo Quick-DNA HMW MagBead Kit on pentagona Seastar Tissue

Testing the Zymo Quick-DNA HMW MagBead Kit with one Cryptasterina pentagona tissue sample in DNA/RNA shield

Sample Prep

  1. Added 260µl of Pro k storage buffer to the 5mg of powder Pro k provided with the kit, then kept on ice
  2. Thawed one PG sample on ice
  3. Prepped two 1.5mL tubes, each with 95µl DNA elution buffer, 95µl Biofluid and Solid Tissue buffer, and 10µl Proteinase K
  4. Took out the piece of tissue, cut it in half on foil, then cut one half in half again. Put one 1/4th piece of tissue in each prepared tube. Placed the rest of the tissue back in the tube with the DNA/RNA shield and re-froze at -20
  5. Pipetted to mix with p200 pipette
  6. Placed in thermomixer, 55 degrees C and shaking at 400rpm. Put in at 3:39pm and planned to go overnight
  7. Samples were taken out of the thermomixer at 9:15am the next day. note: samples weren’t completely broken down, there was some tissue bits left around the bottom of the tubes. PG1 had what I would guess were fat bits floating at the top but those weren’t in PG2
  8. Centrifuged tubes at 10,000 rcf for 1 minute
  9. Tried to transfer only liquid to new 1.5mL tubes, basically failed at that even with the p20 pipette, there was still some tissue, or more likely mucus/lipids that came to the next tube
  10. Centrifuged again now at max rcf for 2 minutes
  11. Transferred what liquid I could without getting any chunks to new 1.5mL tubes again. PG1 had a few floating mucus/lipid bits that I could not avoid pipetting. PG2 had a top orange layer in the liquid that I also transferred

DNA Purification

  1. Added 400µl of Quick-DNA MagBinding Buffer to each sample and pipetted to mix this addition seemed to remove/dissolve any mucus-y-ness that was left in the samples ¯\_(ツ)_/¯
  2. Gently shook/swirled MagBinding Beads to make sure they were resuspended
  3. Added 33µl MagBinding Beads to each sample, making sure to shake the bottle of beads before aspirating from it each time
  4. Pipetted to mis the beads inside the sample tubes
  5. Placed on the shaker for 15 minutes, the protocol said 1100 rpm, but I thought that was really fast and I thought the tubes were going to come of the shaker, so I did 740 rpm. However, I came back after 15 minutes and saw that some of the beads had settled so I switched the tubes to the thermomixer at 23 degrees C and 12,000 rpm for 10 minutes. This time the beads were resuspended
  6. Placed the tubes on the magnet rack an waited until the beads went to the magnet. note: the liquid never went clear, but it was yellowish so I thought it was the tissue remnant not the beads because the beads are opaque black
  7. Carefully removed and discarded supernatant
  8. Took tubes of the magnet and added 100µl DNA Elution buffer to each sample and pipetted to mix note: PG1 had what was like a clump of beads that wouldn’t really breakup, but I was being gentle and din’t try very hard also
  9. Added 500µl of Quick-DNA MagBinding Buffer to each tube and pipetted up and down to mix (still saw the clump in PG1)
  10. Placed on the thermomixer 1200rpm for 10 min at 23 degrees C
  11. Took out samples and placed on the magnet rack
  12. When the supernatant was clear, removed and discarded it from each tube
  13. Took samples off the magnet and resuspended the beads in 500µl DNA Pre-wash Buffer and pipetted 10 times to mix
  14. Placed the tubes back on the magnet stand and removed the supernatant when it was clear
  15. Took the tubes off the magnet and resuspended the beads in 900µl g-DNA Wash Buffer and pipetted 10 times to mix
  16. Removed entire volume of each sample including the beads and placed into new 1.5mL tubes
  17. Placed tubes on magnet and removed the supernatant when it was clear
  18. Took tubes off the magnet and resuspended the beads in 900µl g-DNA Wash Buffer and pipetted 10 times to mix
  19. Removed the entire volume of each sample including the beads and put into new 1.5mL tubes
  20. Placed tubes on magnet stand and removed the supernatant when it was clear, used a smaller pipette to get rid of any excess liquid I could see in the tube
  21. Left the tubes on the magnet stand with their tops open for ~25 minutes. The top of the beads were nice and matte but the bottom was still a little bit damp looking, but no beads were looking glossy anymore. Did not want to over dry, at first I set a timer for only 15 minutes but the beads were very wet then still
  22. Resuspended the beads in 50µl DNA elution buffer from the kit and pipetted at least 20 times to mix
  23. Placed the tubes on the thermomixer at 23 degrees C 1200 rpm shaking at 11:32am.
  24. Tubes were taken off the shaker at 4:15pm and placed on the magnet rack
  25. The clear supernatant was taken and SAVED as the DNA! 1µl was used for Qubit and Tapestation each, the rest was put into the -20 freezer

Broad Range Qubit:
Standard 1: 200 RFU
Standard 2: 23234 RFU
PG1: 96.2ng/µl, 95.8ng/µl, average: 96ng/µl
PG2: 127ng/µl, 126ng/µl, averageL 126.5ng/µl

Genomic DNA TapeStation:

1 2

full report

Written on October 31, 2019