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