Difference between revisions of "BBa K731700 and BBa K731710 measurements"
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'''BEFORE STARTING:'''
 
'''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).
 
* 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).
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'''CELLS GROWTH AND SAMPLES PREPARATION (starting from a glycerol stock):'''
 
'''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)
 
* 100ul of cells (i.e. BL21(DE3) pLysS) tranformed with the platform (control or sample) in 10 ml fresh Luria Bertani medium (LB)
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NOTE2:Four glycerol stocks for both control and sample will give you a good level of significance
 
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.)
  
−
*grow the cultures at 37°C in the termoshaker until OD ~ 0.4 (the OD 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.
−
*(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.
+
**LB = 10ml-x where LB are the ml of LB you need to add to x to obtain OD 0.2.
−
**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.
 
*grow 37°C in the termoshaker until OD ~ 0.6.
 
*induce with 0.5mM IPTG.
 
*induce with 0.5mM IPTG.

Revision as of 08:40, 22 September 2012

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

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 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


Measurements of part K731700 and K731710