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.
- 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[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.