Sam:LabNotes/Microbiome-new/2011-4-5

From ZhangLabWiki
Revision as of 00:34, 6 April 2011 by >Sam Chiang (→‎TrypanBlue(TB) staining)
Jump to navigation Jump to search

Single E.coli cell micromanipulation new test on TrypenBlue stained E.coil

Background

  • From previous test on 03-31-11, we learned how to set up some basic conditions for targeting E.colis
    • Use cover slide to keep sample from evaporation.
    • Able to target E.coli cell without any staining.
    • Still can see many debris >= the size of E.coli. Hard to define

Objective

  • Test Trrypen BLue stained E.coli
  • Improve glass slide washing using isopropanol
  • Improve E.coli washing using 0.2um filtered 1XPBS.
  • Make new micro-pipette tips with narrower mouth-open between 10~20um (by Andy)

TrypanBlue(TB) staining

  • Use the same E.coli culture from 03-31-11 test
  • E.coli washing at low-speed (4000 rpm, 5min at RT)with 0.2um filtered clean PBS.
    • Use low speed to avoid the precipitation of cell debris.
  • Resuspend the E.coli cells in 1.5mL PBS -> low-speed pelleting -> Discard supernatant
  • Repeat the last step again.
  • Resuspend in 1.5mL PBS (estimated conc. as 10^9/mL).
  • Prepare two 1.7mL tube labeled as TB(E.coli) and TB(Neg)
  • Transfer 10uL E.coli (10^9/mL) culture into the 1.7mL tube. Transfer 10uL PBS in the TB(Neg.) tube.
  • Add 10uL undiluted TrypenBlue into 1.7mL tubes. Mix by repeat pieptting and incubate at RT for about 10min.
  • Add 980uL 1XPBS (estimated conc. became 10^7/mL)
  • Perform anther 1/100 dilution by adding 2uL stained sample in 198uL PBS (estimated conc. became 10^5/mL)
    • since I use low-speed washing in the first step, I lost more E.coli during washing so I start with 10^5/mL for micromanipulation.