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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NEED WHAT?
- In order to analyse terminators through this method is necessary to follow the entire procedure with both the empty platform (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 37°C in the termoshaker until OD ~ 0.4.
- (chill on ice for the whole duration of this point) dilute samples to OD 0.2 in order to have the same OD at the moment of induction.
- x = 10ml*0.2OD/0.4OD ___ 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 OD ~ 0.6.
- induce with 0.5mM 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 10s. 50% amplitude sonication and 30s. of pause).
- 1.5min centrifuge.
- move supernatant from eppendorf to cuvette.
- add 2ml buffer (i.e.: PBS)
- leave the cuvettes O/N in the 4°C fridge
- fluorimetric measurements
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Measurements of part K731700 and K731710
