Sam:LabNotes/Micro-manipulation/2011-4-19

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Revision as of 00:03, 27 April 2011 by >Sam Chiang (→‎Results)
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Single E.coli cell micromanipulation - Glycerol test

Objective

  • Test if effect of adding glycerol on DAPI staining
  • Optimize the ideal glycerol adding concentration to obtain the best bacteria response for micro-manipulation.

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. Resuspend in 1mL PBS.
  3. Filter the cell suspension through 5um-filter.
  4. Pellet at 5min, 8000 rpm. Remove supernatant. Resuspend in 0.5mL PBS.
  5. Prepare the following glycerol solution in H2O (clean H2O left from GA-sequencer)
    1. 0.1%, 1%, and 10% (v/v)
  6. Equal volume replacement for adding glycerol in cell suspension.
    1. Remove 50uL of cell suspension and add 50uL diluted glycerol in the suspension.
    2. The new glycerol concentration will be 0.01%, 0.1%, and 1%. I also prepare the positive control which has no glycerol added.
  7. Prepare DAPI working solution (5mg/mL -> 1/5 dilution with H2O to 1mg/mL).
  8. Add DAPI working solution in the E.coli. Add 2uL dye for each of the tubes. Repeat ipetting slowly 10X~20X to mix the dye with sample.
  9. Incubate in on the rocking plate/Eppendorf rocking incubator at RT (25C), 400~500rpm for 15min.

Results

File:042111-noglycerol bf1.png  File:042111-noglycerol dapi200.png

File:042111-0 01glycerol bf1.png  File:042111-0 01glycerol dapi200.png

File:042111-0 1glycerol bf1.png  File:042111-0 1glycerol dapi200.png

File:042111-1glycerol bf1.png  File:042111-1glycerol dapi200.png

Discussion

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