Sam:LabNotes/Microbiome-new/2011-3-31: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Sam Chiang
>Sam Chiang
Line 10: Line 10:
**Innoculate E.coli K-12 into the LB mediua(using 10p tips)
**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)
**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.
**(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.
**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.
**Resuspend the pellet in 1.5mL 1X PBS. Transfer into a 1.7mL tube.

Revision as of 06:38, 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)

become