Bacterial Protein Expression - Test Induction


Required Materials:  

1M IPTG (isopropyl-1-thio-β-D-galactopyranoside)
238 mg of IPTG
900 µl of ddH2O

† Dithiothreitol (DTT) can be used instead of β-mercapto-ethanol
‡ Pyronin Y can be used instead of Bromophenol Blue, or the buffer can be clear

Sample Buffer
62.5 mM Tris-HCl pH 6.8
   2 mM β-mercapto-ethanol (β-ME)†
  10 %  glycerol
   2 %  Sodium Dodecyl Sulfate (SDS)
0.01 %  Bromophenol Blue

**(CAUTION - both β-mercaptoethanol and SDS are hazardous. They should be handled with care inside a ventilated fume hood)**

Protocol:

Day 1
1) Transform a protein expression competent E.coli strain (such as BL21) with the plasmid of interest.
*Remember, pET vectors require BL21 with the DE3 cassette*

2) Spread transformed cells on LB-Agar plates with the proper antibiotic and grow overnight at 37°C.

Day 2
3) Pick two colonies from the overnight LB-Agar plate to start 3 ml overnight LB cultures with the proper antibiotic. Grow at 37°C with shaking.

Day 3
4) Start two fresh 3 ml LB cultures from the overnight LB cultures. Dilute 30 µl of each overnight culture into two different tubes containing 2.70 ml of LB. There will be a total of four tubes, two for each clone. One tube from each clone will be for induction; the other will be a non-induced control.

5) Grow fresh cultures at 37°C with shaking for 1 hour.

6) Add 1-2 mM of IPTG to one of the two tubes for each clone. This will be the induced culture. Do nothing to the second tube for each clone. This will be the un-induced control.

7) Take a 100 µl sample of each tube every hour. A sample can be taken immediately preceding IPTG addition as a 0 time point, but is not necessary.

8) Spin the 100 µl sample in a tabletop centrifuge at 3,500 x g for 2 minutes to pellet the bacteria.

9) Remove the supernatant and re-suspend the bacterial pellet in 50 µl of 2X Sample Buffer.

10) Heat the re-suspended sample at 70°C for 10 minutes.

11) Spin the sample at full-speed in a tabletop centrifuge for 3 minutes in order to pellet the genomic DNA.

 

© 2008 Joseph Roland
www.Cytographica.com