Lidstrom:Hot water extraction

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Overview

  • total time = ~ 1 week
    • 3 rounds of lyophilization; takes ~3 days total. (1 after fast filtration, two after hot water extraction)
  • cell volume requirement:
    • 50mL of of OD ~0.5 per mass spec sample works for 5GB1. For other bacteria with different pool sizes, these numbers vary.

Day 1: Harvest Cells

  • For growing cells, 1-2 doublings suggested, 0.6~0.8 OD600, for my barely utilizing cells, Yanfen suggest 50-100 mL
  1. Label 50 mL falcon tubes
  2. Setup Filtration Equipment
    • Filtration setup with vacuum flask (see pictures below)
    • Filters, Nylaflow, Nylon Membrane Filter 0.2 um, 90 mm, P/N 66603 [Pall Life Sciences]
    • Flat-tipped tweezers
    • Liquid nitrogen in Dewar
  3. Put a new filter in the setup
  4. Start vacuum
  5. Pour ~25 mL sterile filtered water through the filter
  6. Pour from a tube or pipet samples onto the center of the filter. It should take ~30 sec to filter samples
    • Hot Water Extraction 1.jpg
  7. Once sample is filtered. Expose filter and fold into quarters with the tweezers.
    • Hot Water Extraction 2.jpg
  8. Put folded filter into a clean, labelled 50 mL Falcon tube
  9. Close tube and flash freeze in liquid Nitrogen.
  10. Repeat for each sample.
  • Pause Point: Samples can be stored at -80 deg C at this point.
  1. Replace caps on frozen tubes with ones that have holes punched in them.
  2. Lyophilize for 8 hrs or overnight (this can be cut shorter if needed, but we do longer just to be safe)

Day 2: Hot Water Extraction

  1. Preheat hot water baths to 100°C (~1 hr before using). Add additional DI water so it will cover the most of the tubes. °C
    • Put a 9-50 ml tube rack in each water bath
  2. Boil filtered sterile water in the microwave (3 min).
    • Put boiling water in boiling water bath to keep hot while doing the next step
  3. Add 25 mL boiling water to each sample, cap tightly and vortex to mix.
  4. Put tubes in boiling water for 20 min. Make sure the water level in the bath is above the liquid level in the tubes.
  5. Put the tubes on ice for 40 min. Use a deep ice bucket.
  6. Pre-cool centrifuge.
  7. Vortex tubes for 1 min each.
  8. Remove the filter from each tube. Try to keep all liquid in the tube. Use the pipette to remove pockets that are stuck in the folds.
  9. Centrifuge 20 min, 5,000rpm, 4°C
  10. Decant into fresh tubes.
  11. The remaining tubes containing protein and cell debris were centrifuged again, at 4°C, 5000rpm for 20 minutes, and supernatants were removed carefully into the clean tubes.
  12. Flash freeze clean supernatants with liquid Nitrogen.
  • Pause Point: Samples can be stored at -80°C.
  1. Replace caps with caps with holes in the lids.
  2. Lyophilize for ~24-36 hours until dry.

Day 4: Reconstitute #1

  1. Replace caps with ones removed before lyophilization.
  2. Reconstitute sample in 1 mL sterile, filtered ddH2O
  3. Vortex to mix and short spin to collect liquid
  4. Transfer to 1.7 mL Eppendorf tubes
  5. Flash freeze with liquid Nitrogen
  6. Open tubes and cover with parafilm. Puncture holes in parafilm. Put tubes in foam holder
  7. Lyophilize tubes for until dry (~overnight)

Day 5: Reconstitute #2

  1. Reconstitute in 100 uL sterile, filtered ddH2O.
  2. Centrifuge tubes, max speed, 4°C, for x 10 minutes
  3. Filter supernatant with spin column filter (centrifuge until all liquid has passed through the filter)
  4. Pipet sample into vial insert in MS vials with split lids
  5. Sample is ready for LC-injection!