Mathies:Culture in Broth

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E. COLI CULTURE IN BROTH AND QUANTIFICATION

Reagents

Bacteria: E. coli strain K12 and O157

Broth: Tryptic Soy Broth medium (TSB, Hardy Diagnostics K131); Lewis 310 F3, stored 2ml broth per glass tube

Antibiotic: stock Ampicillin solution with a concentration of 100mg/ml for E. coli strain K12; (Spectramycin solution for E. coli strain O157 with spec resistance gene or constantly test contamination in O157 cells without spec resistance gene by PCR)

Protocol

1) Thoroughly clean the hood with 70% ethanol

2) Place the tube of desired cells in the hood to thaw the solution, slightly spin down the cell solution to the bottom of the 500µl tube.

3) Transfer 50µl of the cell solution into a 2ml TSB growing medium

4) Add 2µl of 100mg/ml ampicillin solution into the K12 grow medium (or 2µl of 100mg/ml spectramycin into the O157 grow medium)

5) Do NOT tightly cap the TSB tube so oxygen exchange can be possible, tape the cap and the tube together before putting into the Bioshaker V.BR-36

6) Set the temperature to 37˚C and the speed dial pointing almost vertical

7) Grow cells in above conditions over night

The next day

8) Transfer the grown cells into a 15ml/high-clarity polypropylene conical tube

9) Centrifuge the cells at 3500rpm for 3 minute in eppendorf Centrifuge 5804

10) Discard the supernatant and add 2ml filtered PBS, gently suspend the cell solution

11) Repeat step 9) and 10) two more times

12) Make 10X dilution from the grown cell solution before UV measurement

13) Measure OD600 of the 10X dilution using JASCO V-530 UV/VIS spectrophotometer (Use disposal cuvettes, which are placed at the shelf and purchased from LSA stock room with C# of P2180) Average three OD600 measurement values is highly recommended.

14) Calculate the concentration of the cell solution using calibration curves (Please see the section of “CALIBRATION CURVE” for details)

Contact

or instead, discuss this protocol.

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