Timp:ecoliProtocol

E.coli Treatment Protocols
Includes all protocols for culturing, handling and treatment of E.Coli.

Plate Preparation

 * Materials:
 * 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


 * Procedure:
 * To make approximately 5 plates:
 * 1) 200ml DI water
 * 2) 8 capsules LB Agar
 * 3) Autoclave and let cool
 * 4) Add appropriate anti-biotic (200μL) (Kanamaycin for 111’s, 203’s, Ampicillin for M1’s and both for Oscillator cells)
 * 5) Pour in Petri dishes, let it settle and Para film.
 * 6) Parafilm, and store at 4C.
 * 7) To streak plates, touch single colony with inoculating loop and streak in zig-zag pattern. Leave in warm room (37 C) over night.

Sample Preparation

 * Materials:
 * 14ml Polystyrene Round-Bottom Tube (Falcon 352057)
 * Inoculating loops (Fisherbrand 14-375-103)
 * M9 Media
 * Procedure:
 * 1) Touch single colony with inoculating loop and put it in 5mL of M9 media in round bottom tube.
 * 2) Leave it in the warm room shaker over night.
 * 3) Before the experiment dilute the saturated culture 1:5 and let it sit in the RT shaker for at LEAST 2hrs.
 * 4) 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.
 * 5) Spin it down again and gently re-suspend in M9 with no antibiotics.

Freezing E.coli in Glycerol Solution

 * Materials:
 * Glycerol - Sigma G2025-500mL
 * Magnesium sulfate - Sigma M2643-500g
 * Tris*Cl pH 8
 * 2.0 ml Cyrogenic Vial (Corning-430659)
 * Procedure:
 * 1) Add ingredients in the following concentrations (brackets show values for 50mL):
 * 2) 60% Glycerol (30mL)
 * 3) 0.1M MgSO4 (0.602g)
 * 4) 0.025M Tris *Cl pH 8 (0.197g)
 * 5) Rest is 18 Mohm water (20mL)
 * 6) Mix 1mL of freshly saturated culture with 1mL of glycerol solution in cyro Vial
 * 7) *(an overnight culture will do here)
 * 8) Immediately store vial in -80C freezer

Thawing Glycerol Frozen E.Coli

 * Materials:
 * LB Agar plates (refer to Plate Preparation protocol above)
 * Frozen Cell Culture
 * Inoculating Loop
 * Parafilm
 * Procedure:
 * 1) Scrape out a splinter or two of frozen culture and streak onto plate.
 * 2) After Splinter has thawed, streak with inoculating loop
 * 3) Grow at 37 C overnight. Plate should be inverted at ALL times.
 * 4) Next day examine plate. Plates may be stored wrapped in Parafilm at 4C for less than one month safely.