Sam:LabNotes/Microbiome-new/2011-3-31: Difference between revisions
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>Sam Chiang (Created page with '='''Single E.coli cell isolation using micromanipulation''' ==Background== *') |
>Sam Chiang |
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==Background== | ==Background== | ||
* | *Collabrate with Andy Chen | ||
*Adapt the successful experience of micromanipulation on single human lymphocyte | |||
==Fresh E.coli cells revovery== | |||
*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) 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)== | |||
become |
Revision as of 06:15, 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 revovery
- 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) 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)
become