Sam:LabNotes/Micro-manipulation/2011-4-26

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Revision as of 00:52, 27 April 2011 by >Sam Chiang (→‎Discussion)
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Single E.coli cell micromanipulation - Fresh sample test

Objective

  • I compare DAPI-staining performance on fixed(EtOH) and non-fixed E.coli cells(using the old E.coli sample stored from 03-10-'10 in 4C). The fixed cell still give me bright DAPI signal, but the non-fixed E.coli cell showed very dim single as previous tests I did.
  • I start doubting if the E.coli samples in the non-fixed samples are too old or the DNA were degraded in these cells.
    • When bacteria are dying the DNA will be degraded slowly, however I thought the degradation is too slow to be regarded as a concern.
  • I grew the fresh E.coli cell on 04-25-'11, and will compare fresh cells with old cells on DNA dye staining again.

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:042611-noglycerol bf1.png  File:042611-noglycerol dapi200.png

Discussion

Follow up

  • 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.