IPTG Induction of Ratiometrica in E. coli

Risk Assessment

This protocol is an adaptation of this one: http://www.genetics.wustl.edu/tslab/?page_id=107 and is used to test the induction of the pSPANK promoter with IPTG in E.coli

Preparation

Have 1M IPTG stock solution made up and stored in the fridge.

Agar plates

1) Pick cultures and grow in 2ml LB/AMP (100ug/ml) in a 15ml snap cap tube overnight at 37⁰C at 160RPM.

2) Dilute 1:100 and grow for 3-4 hours in 2ml LB/AMP (100ug/ml) in a 15ml snap cap tube

3) Prepare LB/AMP/IPTG plates with 100ug/ml of Ampicillin and a final concentration of 0.5mM. Also have a couple of normal amp plates ready.

3)Spread 250uL and 25uL onto you IPTG plates. Also spread some on normal amp plates as a control. Allow to grow in incubator.

Liquid cultures

1) Pick cultures and grow in 2ml LB/AMP (100ug/ml) in a 15ml snap cap tube overnight at 37⁰C at 160RPM.

2) Dilute 1:100 and grow for 3-4 hours in 2ml LB/AMP (100ug/ml) in a 15ml snap cap tube

3) In eppendorf tubes, make up 1mL LB/AMP/IPTG solutions with varying IPTG concentrations (2 times the intended concentration, i.e. if the final concentration desired is 1mM IPTG, make a 2mM solution)

4) Take 1ml of the cell culture in the snap cap tubes as uninduced controls; add the 1mL LB/AMP/IPTG into the remaining culture (1mL) in the snap cap tubes; grow for 3-4 hours at 37°C

Notes

LB autoflouresces. Use M9 minimal medium for plates and cultures if quantification of fluorescence (e.g. in plate reader) is needed. It takes approximately twice the amount of time for cells to grow in M9 minimal medium.