Sam:LabNotes/Micro-manipulation/2011-4-19: Difference between revisions
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#Equal volume replacement for adding glycerol in cell suspension. | #Equal volume replacement for adding glycerol in cell suspension. | ||
##Remove 50uL of cell suspension and add 50uL diluted glycerol in the 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%. | ##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). | #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. | #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. |
Revision as of 23:23, 26 April 2011
Single E.coli cell micromanipulation - Glycerol test
Objective
- 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
- 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:04-19-11-Dual stained 2uL Ecoli BF-10X.jpg File:04-19-11-Dual stained 2uL Ecoli DAPI-10X.jpg File:04-19-11-Dual stained 4uL Ecoli BF-10X.jpg File:04-19-11-Dual stained 4uL Ecoli DAPI-10X.jpg