Tuesday, 28 February 2012

Protocol for the Blue White Colony Screening or Selection

Composition of LB Medium:

1.0% Tryptone 0.5% Yeast Extract 1.0% Sodium Chloride (NaCl) pH 7.0

Batch Protocol

Following Protocol makes 1 liter LB agar plates:

Dissolve 10 g Tryptone, 5 g yeast extract, and 10 g NaCl in 950 ml ultra pure water. Adjust the pH of the solution to 7.0 with NaOH. It may not be necessary to adjust pH every time, since pH of LB naturally close to pH 7.0. This might be affected by your reagents. Add 15 g agar. Complete the volume to 1 liter. Autoclave on liquid cycle for 20 minutes at 15 psi. Cool the media to 50 C in a water bath. Add 1 ml of X-Gal. Add 1 ml of 100 mM IPTG Solution. Add antibiotic for selection. Mix well and pour prepared LB agar into plates. Dry opened LB plates at room temperature under UV light for about half an hour.

Individual Plate Protocol

Add 40 µl of the X-Gal Solution (20 mg/ml). Add 10 µl of 100 mM IPTG Solution. Spread evenly on the plate with a sterile spatula. Dry the plate at 37°C. Spread bacteria evenly on the plate. Incubate overnight at 37°C.

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