Bacteria Culture for Protein Expression, written by Rosa Schier

Remember to choose your antibiotics

LB media 1X: weigh 27.5g of 2xLB-broth powder (from Formedium) and adjust up to 1l with ddH2O to dissolve (110g for 4l), Autoclave

TB media: weigh 6.5g of glycerol and 47.6g of terrific broth, phosphate buffered powder (from Formedium) and adjust up to 1l with ddH2O to dissolve (26g glycerol, 190.4g TB powder for 4l), give it to autoclave

Note: We also commonly use AutoTB medium.

Day 1 before the large culture (preculture):

Put 50 ml of LB or TB media (either with the antibiotics already or add 50ul of antibiotics in 50ml, typically our stocks are 1000X) in an 250ml Erlenmeyer (around a flame) + on a tip a colony or streak from the transformation
Shake at 220rpm and 37°C overnight

We are using 50 mL here as we typically innoculate 10-20 mL / 1 L of culture. One will need to scale up accordingly for larger volumes

Day 2 and 3 day of the culture:

Keep a blank of 1ml of LB or TB to measure the density
2l of LB or TB medium in an 5L erlenmeyer + 2ml of antibiotics + 20-40 ml of preculture for every 2 L
Shake at 190rpm and 37°C
Defrost the IPTG stock at 1M
Measure the density of the culture after 2 hours of shaking
Around every 20 min the density should double. This is typically with one antibiotic. It usually is ready to be induced around 2.5 - 3 hrs.
If the density is between 0.6 and 0.8 (keep this number), take a sample of 1ml (-IPTG) for the expression test and add 2ml (for 1mM) or 1ml (for 0.5mM) of IPTG/2l of culture
Put at 18°C and 140rpm overnight or leave at 37°C and 190rmp for 3 hours
Measure the density and take a sample of 1ml (+IPTG)
Centrifuge 20-30 min at 5000xg
Put the supernatant in an erlenmeyer + vikron 30 min => waste
Get the pellet in a 50ml tube and freeze it (-20°C)
Expression test -/+IPTG: spin 3 min at 5000rpm throw away the supernatant and resuspend in 500ul urea at 8M, for the gel you need the density to calculate how much sample you need