Lidstrom:Hot water extraction: Difference between revisions
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[[Lidstrom:Protocols | | Return to [[Lidstrom:Protocols | 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 [[http://www.pall.com/main/laboratory/product.page?id=20053 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. | |||
# Lyophilizer | |||
==Day 2: Hot Water Extraction== |
Revision as of 16:36, 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.
- Lyophilizer