Lidstrom:Hot water extraction: Difference between revisions
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# Pour ~25 mL sterile filtered water through the filter | # 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 | # Pour from a tube or pipet samples onto the center of the filter. It should take ~30 sec to filter samples | ||
#*[[Image:Hot_Water_Extraction_1.jpg |thumb|center]] | |||
# Once sample is filtered. Expose filter and fold into quarters with the tweezers. | # Once sample is filtered. Expose filter and fold into quarters with the tweezers. | ||
#*[[Image:Hot_Water_Extraction_2.jpg |thumb|center]] | |||
# Put folded filter into a clean, labelled 50 mL Falcon tube | # Put folded filter into a clean, labelled 50 mL Falcon tube | ||
# Close tube and flash freeze in liquid Nitrogen. | # Close tube and flash freeze in liquid Nitrogen. | ||
# Repeat for each sample. | # Repeat for each sample. | ||
*Pause Point: Samples can be stored at -80 deg C at this point. | *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. | # 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) | ||
#* [[http://www.labconco.com/product/freezone-45-liter-benchtop-freeze-dry-systems/97| Labconco FreeZone 4.5 -105°C]], vacuum -.008 mbar, temperature -108°C | |||
==Day 2: Hot Water Extraction== | ==Day 2: Hot Water Extraction== | ||
Revision as of 16:48, 5 November 2014
Return to Lidstrom Protocols
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
