E.coli Treatment Protocols
Includes all protocols for culturing, handling and treatment of E.Coli.
- LB-Agar Medium capsules (Bio 101 Systems)
- Ampicillin Ready-made Solution (Sigma A5354-10ML)
- Kanamyacin solution from Kanamyacin Sulfate- (Shelton Scientific IB02120)
- DNAse, RNAse free Distilled Water (Gibco 5288)
- Para film
- To make approximately 5 plates:
- 200ml DI water
- 8 capsules LB Agar
- Autoclave and let cool
- Add appropriate anti-biotic (200μL) (Kanamaycin for 111’s, 203’s, Ampicillin for M1’s and both for Oscillator cells)
- Pour in Petri dishes, let it settle and Para film.
- Parafilm, and store at 4C.
- To streak plates, touch single colony with inoculating loop and streak in zig-zag pattern. Leave in warm room (37 C) over night.
- 14ml Polystyrene Round-Bottom Tube (Falcon 352057)
- Inoculating loops (Fisherbrand 14-375-103)
- M9 Media
- Touch single colony with inoculating loop and put it in 5mL of M9 media in round bottom tube.
- Leave it in the warm room shaker over night.
- Before the experiment dilute the saturated culture 1:5 and let it sit in the RT shaker for at LEAST 2hrs.
- Spin it down at 1.4krpm for 2 min and re-suspend the pellet in M9 with appropriate antibiotic and let it sit in a vial for 15 min.
- Spin it down again and gently re-suspend in M9 with no antibiotics.
Freezing E.coli in Glycerol Solution
- Glycerol - Sigma G2025-500mL
- Magnesium sulfate - Sigma M2643-500g
- Tris*Cl pH 8
- 2.0 ml Cyrogenic Vial (Corning-430659)
- Add ingredients in the following concentrations (brackets show values for 50mL):
- 60% Glycerol (30mL)
- 0.1M MgSO4 (0.602g)
- 0.025M Tris *Cl pH 8 (0.197g)
- Rest is 18 Mohm water (20mL)
- Mix 1mL of freshly saturated culture with 1mL of glycerol solution in cyro Vial
- (an overnight culture will do here)
- Immediately store vial in -80C freezer
Thawing Glycerol Frozen E.Coli
- LB Agar plates (refer to Plate Preparation protocol above)
- Frozen Cell Culture
- Inoculating Loop
- Scrape out a splinter or two of frozen culture and streak onto plate.
- After Splinter has thawed, streak with inoculating loop
- Grow at 37 C overnight. Plate should be inverted at ALL times.
- Next day examine plate. Plates may be stored wrapped in Parafilm at 4C for less than one month safely.