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Bacteria Growth

The document outlines the standard procedure for inducing and freezing BL21 cells expressing GST-fusion proteins. Key steps include: 1) Growing BL21 cells overnight in LB medium with antibiotics at 37°C. 2) Inoculating 6L of fresh LB medium with overnight culture and growing at 37°C until OD600 reaches 0.5-1.0. 3) Inducing protein expression by adding IPTG and reducing temperature to 18°C for overnight shaking. 4) Harvesting and centrifuging cells, resuspending in sonication buffer, and storing in 50mL tubes submerged in liquid nitrogen.

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82 views

Bacteria Growth

The document outlines the standard procedure for inducing and freezing BL21 cells expressing GST-fusion proteins. Key steps include: 1) Growing BL21 cells overnight in LB medium with antibiotics at 37°C. 2) Inoculating 6L of fresh LB medium with overnight culture and growing at 37°C until OD600 reaches 0.5-1.0. 3) Inducing protein expression by adding IPTG and reducing temperature to 18°C for overnight shaking. 4) Harvesting and centrifuging cells, resuspending in sonication buffer, and storing in 50mL tubes submerged in liquid nitrogen.

Uploaded by

dhashrath
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Standard procedure for inducing and freezing BL21 expressing GST-fusion proteins:

1) Grow 2 x 30mL of LB + 30 ug/mL chlor, 200 ug/mL amp (30 uL each of stocks) inoculated
with transformed BL21(Rosetta) colonies from plate overnight at 37°C.

2) Prepare 6 L of autoclaved LB for next day.

3) In morning, add 10 mL of overnight culture per 1 L of LB+ 30 ug/mL chlor, 200 ug/mL amp
(1 mL each of stocks), for 6 L total. Grow ~4 hrs at 37°C with shaking.

4) Double check OD600 is between 0.5 and 1.0.

5) Reduce temperature to 18°C. Induce by adding 0.2 mL of 1M IPTG (final 0.2mM


concentration) to each liter of culture. Spike amp by adding 0.5 mL of stock to 100 ug/mL amp
per liter.

6) Shake cells overnight at 18°C.

7) In morning, harvest cells. Reduce shaker temperature to 4°C to cool cells meanwhile. Our
centrifuge holds 6 x 0.5 L bottles. Fill and balance bottles. Spin down cells at 2700 g for 20
minutes.

8) Add a small amount of bleach to emptied flasks. Decant supernatant of spun cells into the
bleach flasks. Let sit for ~2 minutes. Empty into sink and flush with lots of water.

9) Since our centrifuge bottles hold only 0.5 L, add the remaining 0.5 L of culture to the pelleted
cells and repeat the spin. Decant supernatant as before.

10) Place bottles with cells on ice in square ice bucket. Add 9 mL Sonicator Buffer (1M NaCl, 25
mM Tris pH 8.0) per liter of cell culture, using spatula and pipettor.

11) Store resuspended cells by placing in 2 x 50mL tubes (labeled), submerge in liquid nitrogen,
place in labeled box in -80 freezer.

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