Sam:LabNotes/Microbiome-new/2011-3-31: Difference between revisions
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='''Single E.coli cell isolation using micromanipulation''' | ='''Single E.coli cell isolation using micromanipulation'''= | ||
==Background== | ==Background== | ||
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**Add 2uL E.colu culture(10^9 CFU/mL) with 200uL 10X dye (SYBR / DAPI). Incubate at 37C on the shaker for >30min. | **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. | **Dilute the stained cell from 10^7 CFU/mL to 10^5 CFU/mL with 1X PBS. | ||
==E.coli cell micromanipulation test== | |||
*Test of non-stained cell at BF, 10X condition | |||
**Able to target (see) cells with a drop (8~10uL) of 10^4 CFU/mL ~ 10^5 CFU/mL diluted E.coli cells | |||
**10^5 CFU/mL is still a little bit too dense for targeting a single cells. 10^4 CFU/mL is probably the best density. | |||
*Challenges: | |||
**E.coli cell is floating around and difficult to locate | |||
**Contaminaiton of debris (some of them have size larger than an E.coli cell) | |||
**Not able to tell if the target is E.coli or dust or debris. | |||
**Fluorescent stained E.coli need to observed | |||
*Lessons learned: |
Revision as of 07:14, 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.
E.coli cell micromanipulation test
- Test of non-stained cell at BF, 10X condition
- Able to target (see) cells with a drop (8~10uL) of 10^4 CFU/mL ~ 10^5 CFU/mL diluted E.coli cells
- 10^5 CFU/mL is still a little bit too dense for targeting a single cells. 10^4 CFU/mL is probably the best density.
- Challenges:
- E.coli cell is floating around and difficult to locate
- Contaminaiton of debris (some of them have size larger than an E.coli cell)
- Not able to tell if the target is E.coli or dust or debris.
- Fluorescent stained E.coli need to observed
- Lessons learned: