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Induction of Protein Expression in E.coli

 

The night before expression:

Inoculate 100mL 2XYT culture with one colony (or scraping of glycerol stock) of your construct of interest transformed into BL21 E.coli.  Don’t forget to add antibiotic1:1000.  Shake cultures o/n at 37°C overnight.

 

In the morning:

Inoculate 1L of 2XYT with 10 mL of your overnight culture.  Remember your antibiotic. 

 

Shake cultures at 37°C for approximately 2.5 hours, then check OD600 of culture.

 

Grow culture to an OD600 of 0.8.  Take a 1mL sample of culture: this is your “uninduced” sample.  Pellet the bacteria, remove the supernatant and resusupend pellet in 200mL of 2X SDS-PAGE sample buffer.  Freeze at -20°C until ready for gel.

 

For expression of Xenopus proteins, cool 1L culture in an icebath to 20°C using a thermometer cleaned with ethanol to monitor temperature.

 

Induce protein expression with IPTG (final concentration 1mM for pET vectors, 0.4mM for pGEX vectors). 

 

Shake cultures at 20°C for 3-5 hours.  Take a 1mL sample of induced culture.  This is the “induced” sample.  Pellet as before, and add sample buffer. 

 

Spin down the rest of the cultures in the SLA-3000 rotor using the 500mL bottles.  Spin for 15 minutes at 4000rpm, 4°C.  Pour media off pelleted cells.  Resuspend pellets in 10-20mL of cold PBS.  Pool resuspended pellets (if appropriate), transfer to 50mL conical tubes and pellet cells once more using SLA-600 rotor.  Spin for 15 minutes, 4°C, at 4000 rpm.  Pour off supernatant and freeze pellets at -20°C.

 

 

This page last modified 08/11/2011

The Stukenberg Lab and the Burke Lab are in the Department of Biochemistry and Molecular Genetics at the University of Virginia