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Sam:LabNotes/Micro-manipulation/2011-4-19
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='''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:042111-noglycerol_bf1.png|400px]] [[File:042111-noglycerol_dapi200.png|400px]] [[File:042111-0_01glycerol_bf1.png|400px]] [[File:042111-0_01glycerol_dapi200.png|400px]] [[File:042111-0_1glycerol_bf1.png|400px]] [[File:042111-0_1glycerol_dapi200.png|400px]] [[File:042111-1glycerol_bf1.png|400px]] [[File:042111-1glycerol_dapi200.png|400px]] ==Discussion== *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== *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.
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