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
become
**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.