Sediment Extraction Chemical Protocol Optimization Round 3

Objective:

This is the third round of experimentation on this project with the goal of further improving the method of extraction on marine sediments with high humic contents.

Sample information

samples taken during the July 2021 trip to bodega bay were used on both kit and chemical extractions sample notation is as follows:

Sample | chem notation | kit notation| storage condition| ——-|—————|————-|—– SAM_001| S19| S25| ZYMO SAM_002| S20 | S26 | FROZEN SAM_006 | S21 |S27 | ZYMO SAM_007 | S22 |S28 |FROZEN SAM_011 | S23 | S29 | ZYMO SAM_012 | S24 | S30 |FROZEN ————————————————-

Chemical extraction Protocol

Sample preparation

  1. Thaw samples over ice
    • a) add 200μL sample to a pre-filled bead beating tube (lysis matrix B, 0.1mm silicate bead)( approx. 0.2g)
    • b) Add 500ul of a 0.04M solution Sodium Hexametaphosphate over ice, pipette up and down to mix
  2. Lysis
    • a) add 500 μL lysis Solution I, gently pipette up and down to mix
    • b) Flash Freeze sample at -80C ( no liquid nitrogen available)
    • c)Thaw sample
    • d) Bead beat for 30s
    • e) Incubate at 50°C in for 1 hour, using the thermomixer
    • f) Flash Freeze sample in liquid nitrogen or Dry-ice
    • g) Thaw sample and vortex for 10 s
    • h) Incubate at 50°C in for 1 hour, using the thermomixer
    • i) Add 500 μL Lysis II
    • j) Flash Freeze sample in liquid nitrogen or Dry-ice
    • k) Thaw sample
    • l) vortex for 10 s
    • m) Incubate at 65°C in for 1 hour, using the thermomixer
  3. Purification
    • a) Centrifuge 15 minutes at 10,000xg and 4°C.
    • b) Transfer supernatant to a clean microcentrifuge tube.
    • c) Add 1 volume of CI vortex at max speed for 30 s to ensure sample is well combined
    • d) Centrifuge for 10 minutes at 10,000xg and 4°C
    • e) Transfer aqueous supernatant to clean microcentrifuge tube
    • f) Repeat c-e for 2 cycles ( 3 rounds total) on third round use PCI rather than CI
  4. Precipitation
    • a) Add LPA (20μL/ml final concentration) to sample and homogenize.
    • b) Homogenize sample with 0.1 volumes of 5 M NaCl solution followed by 1.5 volumes of isopropanol using a vortex, ensure samples look well combined
    • c) Incubate in the dark at -20°C overnight. (minimum two hours)
    • d) Centrifuge at 1400xg for 30 minutes at 4°C then pipette off the supernatant to leave the pellet, supernatant can be discarded
    • e) To remove isopropanol, pellets precipitated by isopropanol-containing solution are washed 125μL of 70% ethanol
    • f)centrifuge for 10 min at 14,000×g,
    • g) pipette off excess sample following centrifugation then dry sample using the eppendorf Vacufuge on the Dessication-Aqueous setting for 10-15 minutes.
    • h) repeat e-g to clean sample twice.
    • i)Dissolve pellet in 200μL of nucleotide free water. vortex to dissolve.
  5. Clean-up Fraction sample for DNA and RNA (100μl/ tube). then run samples through the Norgen DNA/RNA Clean and concentrator kit according to the manufacturers protocol, with the exception of dual elutions of half volume into the same tube to increase yield.

Kit Extraction protocol

  1. Samples were defrosted on ice and vortexed to mix well.
  2. 200ul (0.2g) of sediment were added to the zymobiomics lysing matrix tubes with 500ul of DNA/RNA shield
  3. Samples were bead beat at 6.5 m/s for 1 minute with a 5 minute rest for 5 cycles. Note: at this point samples were frozen at -80 for 24 hours before proceeeding to step 3
  4. samples were centrifuged at 10,000 rpm for 30 seconds at room temperature
  5. 300ul of supernatant was transfered to a nuclease free tube where 600ul of lysis buffer was added and pipetted up and down to mix.
  6. the total volume was transferred to the yellow spin column and centrifuged at 10,000rpm for 30 seconds.
  7. the spin column was placed in a new collection tube and set aside
  8. 900ul of 100% EtOH was added to the eluate and pipetted up and down to mix
  9. 1/2 this mixture was tranferred to the green spin column and centrifuged at 10,000rpm for 30 seconds; the eluate is discarded
  10. step 9 was repeated a second time to transfer the other half of the ETOH eluate mixture

  11. 400ul of DNA/RNA wash Buffer was added to the column and centrifuged at 10,000 rpm for 30 seconds before adding 80ul of DNAse Mix ( 75ul DNAse prep buffer and 5 ul of DNAse I/ sample) were added to the spin column and set aside to incubate for 15 minutes

  12. Both Green and yellow (DNA and RNA) columns were processed together after the 15 minute incubation period as follows:
  13. 400ul of DNA/RNA prep Buffer was added to the column and centrifuged at 10,000 rpm for 30 seconds
  14. 700ul of DNA/RNA prep Buffer was added to the column and centrifuged at 10,000 rpm for 30 seconds
  15. 400ul of DNA/RNA wash Buffer was added to the column and centrifuged at 10,000 rpm for 30 seconds

  16. spin column was transfered to a clean 1.5ml microcentrifuge tube and 50ul of of Nuclease free water was added to the column and incubated for 5 minutes before centrifuging at 10,000 rpm for 30 seconds for two rounds ( final elution volume 100 ul)

  17. HRC filters (white column) were prepped by adding 600 ul of HRC Prep solutions and centrifuging at 8,000 rpm for 3 minutes
  18. All eluted samples were added onto HRC filters in new 1.5ml tubes and centrifuged at 16,000 rpm for 3 minutes.
  19. samples were frozen at -80C for later down stream processing.
Written on November 30, 2021