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.

Gene expression of the lac operon using IPTG

Purpose:

To show how genes can be regulated by using the lac operon as an example.

Materials:

BL21 (DE3) + egfp (un-induced)
3 agar plates with Kanamycin
0.5 mM IPTG

Methods:
Plate 2 kanamycin plates with un-induced BL21(DE3) + egfp.
Label plates no IPTG and IPTG
Place in 37°C incubator overnight
To turn gene on for expression
Plate 1: Do not add anything
Plate 2: Using a sterile pipette tip, add 8 µL 0.5 mM IPTG per colony.
Allow IPTG to soak in
Incubate at 37°C overnight
Place the plate under UV light to see bacteria glow.
Calculation: To make 0.5 mM IPTG, mix 50 µL 1.0 M IPTG + 50 µL LB broth
This would be a cool extension of this lab that might be more relatable to the students.

Lab by Ingrid Coulson,
9th grade Honors Biology teacher
Broughton High School, Raleigh, NC