Sam:LabNotes/Microbiome-new/2011-3-31: Difference between revisions
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>Sam Chiang |
>Sam Chiang |
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==E.coli stain (SYBR-green / DAPI)== | ==E.coli stain (SYBR-green / DAPI)== | ||
*Prepare dilued staining dye | |||
**Dilute SYBR(200X) into 10X by 1/20 dilution in a 1.7mL tube wrapped in foil | |||
**Thaw DAPI(10,000X)stock solution. Dilute DAPI into 10X by 1/1000 dilution in a 1.7mL tube wrapped in foil. | |||
*Cell staining | |||
**Add 2uL E.colu culture(10^9 CFU/mL) with 200uL 10X dye (SYBR / DAPI). Incubate at 37C on the shaker for >30min. | |||
**Dilute the stained cell from 10^7 CFU/mL to 10^5 CFU/mL with 1X PBS. |
Revision as of 06:47, 2 April 2011
=Single E.coli cell isolation using micromanipulation
Background
- Collabrate with Andy Chen
- Adapt the successful experience of micromanipulation on single human lymphocyte
Fresh E.coli cells recovery
- Recovery fresh E.coli K-12 cell from glycerol stock (-80C)
- Prepare 10mL pre-warmed LB in 10mL culture tube (x4)
- Innoculate E.coli K-12 into the LB mediua(using 10p tips)
- Cap the tube. Vortexing briefly. Incubate at 37C over night (3:00PM~9:00AM next day)
- (The medium look cloudy in all of four tubes)
- Washing cells
- Pellet the cell by centrifuging.
- Centrifuge the 10mL culture tubes at 3000rpm, 4C, 10min. Discard supernatant.
- Resuspend the pellet in 1.5mL 1X PBS. Transfer into a 1.7mL tube.
- Centrifuge the tube at 8000g, RT, 3min. Discard supernatant.
- Resuspend the pellet in 1.0mL 1X PBS.
- Repeat the last washing step again (Two times PBS washing in total).
- Based on the culture cloudy extent, I estimate it's about 10^9 CFU/mL
E.coli stain (SYBR-green / DAPI)
- Prepare dilued staining dye
- Dilute SYBR(200X) into 10X by 1/20 dilution in a 1.7mL tube wrapped in foil
- Thaw DAPI(10,000X)stock solution. Dilute DAPI into 10X by 1/1000 dilution in a 1.7mL tube wrapped in foil.
- Cell staining
- Add 2uL E.colu culture(10^9 CFU/mL) with 200uL 10X dye (SYBR / DAPI). Incubate at 37C on the shaker for >30min.
- Dilute the stained cell from 10^7 CFU/mL to 10^5 CFU/mL with 1X PBS.