Sam:LabNotes/Micro-manipulation/2011-4-26: Difference between revisions
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>Sam Chiang |
>Sam Chiang |
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==Discussion== | ==Discussion== | ||
==Follow up== | ==Follow up== |
Revision as of 00:52, 27 April 2011
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.
- Pellet at 5min, 8000 rpm. Remove supernatant. Wash 1X in 1mL PBS(0.2um-filtered).
- Resuspend in 1mL PBS.
- Filter the cell suspension through 5um-filter.
- Pellet at 5min, 8000 rpm. Remove supernatant. Resuspend in 0.5mL PBS.
- Prepare the following glycerol solution in H2O (clean H2O left from GA-sequencer)
- 0.1%, 1%, and 10% (v/v)
- Equal volume replacement for adding glycerol in cell suspension.
- Remove 50uL of cell suspension and add 50uL diluted glycerol in the suspension.
- The new glycerol concentration will be 0.01%, 0.1%, and 1%. I also prepare the positive control which has no glycerol added.
- Prepare DAPI working solution (5mg/mL -> 1/5 dilution with H2O to 1mg/mL).
- 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.
- 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.