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

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Single E.coli cell micromanipulation - Glycerol test[edit]

Objective[edit]

  • 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[edit]

  • 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[edit]

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[edit]

  • No significant effect on DAPI-staining performance after adding different diluted glycerol.
    • Different brightness on DAPI is primarily due to the microscopy contrast setting.
  • Surprisingly, the sample added with glycerol showed a much better response to micro-pipetting sucking.
    • Our original idea is to use glycerol to slow down the floating mobility of cell during the sucking procedure.
  • Adding glycerol will be added in our standard procedure of sample preparation. However I need to optimize the glycerol concentration with Andy and check which concentration works best for isolation.

Follow up[edit]

  • By using the sample prepared today, Andy extracted another four single E.coli cell and deposited them in PCR tubes. I keep them in 4C fridge waiting for validation.
  • After discussion with Dr. Zhang, I noticed that we should use non-fixed cell instead of the EtOH-fixed cell for isolation.
    • I will test if the current DAPI-staining protocol could work on non-fixed E.coli samples.