Sam:LabNotes/Micro-manipulation/2011-4-18

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Revision as of 00:22, 22 April 2011 by >Sam Chiang (→‎Results)
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Single E.coli cell micromanipulation test on 04-18-'11

Objective

  • Test the dual staining (TryphanBlue plus DAPI) protocol
    • If it works the TryphanBlue staining can help Andy to locate E.coli cell even easier.

Procedures

  • Starting with EtOH fixed cell (stored in -80C) x2
This cell are about 100uL of OD600=1.0 E.coli, fixed in 500uL 100% EtOH for 3hr.
  1. Pellet at 5min, 8000 rpm. Remove supernatant. Wash 1X in 1mL PBS(0.2um-filtered).
  2. Prepare TryphanBlue working solution (0.5mL 0.4% TryphanBlue + 0.3mL PBS)
  3. Resuspend washed cells 100uL TryphanBlue working sol. Repeat pipetting several times.
  4. Incubate in the Eppendorf rocking incubator at RT (25C), 500rpm for 10min.
  5. Pellet at 5min, 8000 rpm. Remove supernatant. Resuspend in 1mL PBS(0.2um-filtered).
  6. Filter the cell suspension through 5um-filter.
  7. Pellet at 5min, 8000 rpm. Remove supernatant.
  8. Resuspend in 0.5mL PBS(0.2um-filtered).Transfer sample in a 1.7mL amber tube.
  9. Prepare DAPI working solution (5mg/mL -> 1/5 dilution with H2O to 1mg/mL).
  10. Add DAPI working solution in the E.coli
    1. Add 2uL dye for the tube labeled as "2". Add 4uL dye for the tube labeled as "4".
  11. Incubate in on the rocking plate/Eppendorf rocking incubator at RT (25C), 500rpm for 15min.

Results

File:04-19-11-Dual stained 2uL Ecoli BF-10X.jpg  File:04-19-11-Dual stained 2uL Ecoli DAPI-10X.jpg

File:04-19-11-Dual stained 4uL Ecoli BF-10X.jpg  File:04-19-11-Dual stained 4uL Ecoli DAPI-10X.jpg

Discussion

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