Sam:LabNotes/Micro-manipulation/2011-4-15

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Single E.coli cell micromanipulation test on 04-15-'11

Objective

  • Further improve the DAPI staining protocol

Experiment design

  • Test the following conditions:
  1. The effect of filtering the fixed E.coli (by 5um-filter)
  2. Washing and resuspend in PBTw to remove background.
  3. Using higher amount of DAPI dye.

Procedures

Test of pre-filtering the EtOH-fixed samples

  • 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. Wash 1X in 1mL PBS(0.2um-filtered).
    1. One of the samples was filtered through 5um-filter, and labeled as "F".
    2. The other one was labeled as "R" following the regular procedure.
  2. Resuspend in 0.5 mL PBS(0.2um-filtered). Transfer sample in a 1.7mL amber tube.
  3. Prepare DAPI working solution (5mg/mL -> 1/5 dilution with H2O to 1mg/mL).
  4. Add DAPI working solution in the E.coli
    1. Add 1uL dye for "R" tube. Add 4uL dye for the "F" tube.
  5. Incubate in on the rocking plate/Eppendorf rocking incubator at RT (25C), 400rpm for 15min.

Test of adding final washing with PBTw

Results

Comparisons of different conditions on DAPI-stained E.coli
 
File:Sam 041411-DAPI ecoli 10X-BF-annoated.png

File:Sam 041411-DAPI ecoli 10X-DAPI-annoated.png

Discussion

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