Blue-White Screening & Protocols

Blue-white screening is a rapid and efficient technique for the identification of recombinant bacteria. It relies on the activity of β-galactosidase, an enzyme occurring in E. coli, which cleaves lactose into glucose and galactose.

Materials

1. X-Gal

2. Dimethylformamide (DMF) and dH2O

3. Isopropyl β-D-1-thiogalactopyranoside, IPTG

4. Screening antibiotic of choice

5. Agar media Plates

Protocol

For Preparation of X-Gal and IPTG see Solutions and Stocks.

LB plates preparation

X-Gal and IPTG can be incorporated into agar media before pouring into plates or added onto pre-made plates.

1. Prepare 20 mg/ml X-Gal solution in DMF. For reduced DMF toxicity in media, you can alternatively make a 100 mg/ml X-Gal solution in DMF (this concentration is only stable at -20°C for ~1 week).

2. Prepare 100mM IPTG solution in dH2O (or dilute from 1M IPTG Stock Solution).

3. Cool autoclaved growth media agar to 50°C.

4. Add 10 µl X-Gal Solution (20 mg/ml) per 1 mL of Media or 2 µl X-Gal Solution (100 mg/ml) per 1 mL of Media.

5. Add 10 µl IPTG (100mM) per 1 mL of Media for a final concentration of 1 mM.

6. Add screening antibiotic of choice (Ampicillin, Kanamycin, Carbenicillin, etc).

7. Pour plates and allow to cool to room temperature (usually at least 30 minutes) before use.

8. Spread transformed competent cells as desired.

Note: Blue/White Selection plates are generally stable for only 1 week if stored at 4°C in clear sleeves but may be stored in the dark (or a dark sleeve) at 4°C for up to 1 month.