Protocol for induction test at 30 degrees C
This is a generic protocol for doing an expression test for induction conditions. Additional conditions not listed below (such as other temperatures, and IPTG concentrations, etc.) can be accommodated.
Luke Wheeler
Day #1)
1) Pick colonies from a plate of transformed Rosetta™(DE3)pLysS Competent Cells transformed with expression plasmid
to start overnight 10mL culture in LB+Ampicillin+Chloramphenicol (grow o/n at 37C w/ shaking @ 250rpm)
Day #2)
2) Use 1:200 volume of overnight culture to start new 20mL cultures in LB+Amp+Cam (Amp and Cam are 1000x)
3) Grow cultures to log phase (trace growth curve by monitoring OD600) at 37C.
4) Split the log phase cultures 50:50 into new tubes.
5) Induce one of the duplicates for each protein with 1mM IPTG (1000x stock), leave the other uninduced. Optionally,
the culture can also be spiked with 0.2% glucose at the time of induction.
6) Lower temperature to 30C and continue growth for ~4hrs
7) Pellet 1.5mL of each culture by centrifugation (10000 x g for 5 min). For same day experiment, proceed to step 9. For
o/n proceed to step 8 first.
8) Store pellets at -20C.
Day #2/3)
9) Lyse cells in B-PER lysis solution by vortexing (add ~500uL B-PER with ~0.25uL each of DNase1 and Lysozyme).
10) Spin lysate at maximum speed in table-top centrifuge for ~10 minutes to pellet cell debris.
11) Prepare a mixture of Lamelli buffer and 2-mercapto-ethanol (loading buffer for SDS PAGE).
12) Prepare a series of dilutions of the supernatant from step 10 in the Lamelli buffer from step 11 (i.e 2uL, 5uL, 10uL, and 15uL
into 15uL of the buffer). Heat the samples at 95 degrees C for 5-10 minutes then load 15uL of each of these dilutions onto 12%
Tris,HCl acrylamide gel. Run gel with SDS PAGE buffer. Stain gel to visualize protein bands and compare between induced and
un-induced samples.