Lidstrom:Hot water extraction
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- 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
- Label 50 mL falcon tubes
- 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
- Put a new filter in the setup
- Start vacuum
- Pour ~25 mL sterile filtered water through the filter
- Pour from a tube or pipet samples onto the center of the filter. It should take ~30 sec to filter samples
- Once sample is filtered. Expose filter and fold into quarters with the tweezers.
- Put folded filter into a clean, labelled 50 mL Falcon tube
- Close tube and flash freeze in liquid Nitrogen.
- Repeat for each sample.
- Pause Point: Samples can be stored at -80 deg C at this point.
- Replace caps on frozen tubes with ones that have holes punched in them.
- Lyophilize for 8 hrs or overnight (this can be cut shorter if needed, but we do longer just to be safe)
- [Labconco FreeZone 4.5 -105°C], vacuum -.008 mbar, temperature -108°C
Day 2: Hot Water Extraction
- 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
- Boil filtered sterile water in the microwave (3 min).
- Put boiling water in boiling water bath to keep hot while doing the next step
- Add 25 mL boiling water to each sample, cap tightly and vortex to mix.
- Put tubes in boiling water for 20 min. Make sure the water level in the bath is above the liquid level in the tubes.
- Put the tubes on ice for 40 min. Use a deep ice bucket.
- Pre-cool centrifuge.
- Vortex tubes for 1 min each.
- 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.
- Centrifuge 20 min, 5,000rpm, 4°C
- Decant into fresh tubes.
- 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.
- Flash freeze clean supernatants with liquid Nitrogen.
- Pause Point: Samples can be stored at -80°C.
- Replace caps with caps with holes in the lids.
- Lyophilize for ~24-36 hours until dry.
Day 4: Reconstitute #1
- Replace caps with ones removed before lyophilization.
- Reconstitute sample in 1 mL sterile, filtered ddH2O
- Vortex to mix and short spin to collect liquid
- Transfer to 1.7 mL Eppendorf tubes
- Flash freeze with liquid Nitrogen
- Open tubes and cover with parafilm. Puncture holes in parafilm. Put tubes in foam holder
- Lyophilize tubes for until dry (~overnight)
Day 5: Reconstitute #2
- Reconstitute in 100 uL sterile, filtered ddH2O.
- Centrifuge tubes, max speed, 4°C, for x 10 minutes
- Filter supernatant with spin column filter (centrifuge until all liquid has passed through the filter)
- Pipet sample into vial insert in MS vials with split lids
- Sample is ready for LC-injection!