Hello world
In this page we are really proud to introduce you the protocol we (Giacomo and Anna) developed for the characterization transcriptional terminators effects on gene expression. (Tested on BBa_J64997)
The develop of this protocolcost us not only sleepless night but also sunny and beautiful weekend of trekking so take a hot cup of tea and read it all.
IN VIVO ANALYSIS
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BEFORE STARTING:
- In order to analyse terminators through this method is necessary to follow the entire procedure with both the platform without intervening terminator (control) and the platform with terminator (sample).
CELLS GROWTH AND SAMPLES PREPARATION (starting from a glycerol stock):
- 100ul of cells (i.e. BL21(DE3) pLysS) tranformed with the platform (control or sample) in 10 ml fresh Luria Bertani medium (LB)
NOTE1:Antibiotic are at concentration of 0.1mg/ml ampicillin and 0.035mg/ml cloranphenicol
NOTE2:Four glycerol stocks for both control and sample will give you a good level of significance
- Grow the cultures at 37°C in the termoshaker until optical density at 600nm (O.D.) ~ 0.4 (the O.D. at which you stop the sample is not important until it is major than 0.2.)
- (chill on ice for the whole duration of this point) Dilute samples to OD 0.2 in order to have the same O.D. at the moment of induction.
- x = 10ml*0.2 O.D./0.4 O.D. (where x are the ml of culture you need to take from the culture in the termoshaker)
- LB = 10ml-x (where LB are the ml of LB you need to add to x to obtain OD 0.2)
- Grow 37°C in the termoshaker until O.D. ~ 0.6.
- Induce with 0.5mM Isopropyl β-D-1-thiogalactopyranoside (IPTG).
- 3 hours induction at 37°C in the termoshaker.
- Chill falcon on ice
- Move 1ml of induced culture to an eppendorf.
- Sonication (3 repetition of 10 s 50% amplitude sonication and 30 s of pause).
- 1.5 min centrifuge (4000rpm).
- Move supernatant from eppendorf to cuvette.
- Add 1 mL buffer (i.e.: PBS)
- Leave the cuvettes over night (O.N.) it the 4°C fridge
- Fluorimetric measurements
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File:Wls K731710 520V.jp
