Large grow peT - tailspike in BL21

E. coil__________ _________chp

ON= 1 colony into ~200 ml. LB + 100ug/ml in a 500ml baffle flask from bottle of LB amp ( 1ml 50 mg/ml )

Amp grow 37⁰C shaker Overnight

Step 1 To grow cells to a high enough concentration to make a lots of protein:

• add 20 ml. E. coil BL21 with peT-tsp ON to 4 x 1L of LB in baffle with 100ug/ml Amp (Stock is 50mg/ml add 2ml /liter)

  place at 39⁰C In talking to Sean at the phage meeting he has better yield if cells are grown at 39⁰C then shifted to lower temperatures)

• grow to about OD₆₀₀ = 0.4 to ~ 4 x 10⁸ cells/ml (as long as the OD₆₀₀ is below 1 proceed)

Step 2 To turn on the protein production

• Induce culture with IPTG 0.5mM (stock usually is 1M, 0.5 ml /liter
• Shift to the appropriate temperature 30⁰C 3 hr
• (18⁰C) grow for 4-5 hours.

Cool by adding ice and draining water.

Step 3 Take OD₆₀₀ 1/10 (OD₆₀₀ over 1 are on reliable )

Step 4 Concentrate cells spin culture - 1 liter bottle Sorger's cfg 4K 15 min.

Step 5 Resuspend pellet in Buffer B ( 50 mM tris 25 mM NaCl 2 mM EDTA ) pH 8 with
40mM octly- glucoside 292 X 0.4 X vol

Take a 100 ul sample to check later. in to 1.5 ml epi tube.

Step 6 Freeze -20⁰C