Tokyo/IPTG assay
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== IPTG assay == | == IPTG assay == | ||
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===Purpose: === | ===Purpose: === | ||
<br>To determine the order of the concentration of IPTG necessary for the activation of our lux-lac hybrid promoter in the LacI producing pTrc99A cells. | <br>To determine the order of the concentration of IPTG necessary for the activation of our lux-lac hybrid promoter in the LacI producing pTrc99A cells. | ||
Revision as of 00:15, 22 October 2007
Contents |
IPTG assay
Purpose:
To determine the order of the concentration of IPTG necessary for the activation of our lux-lac hybrid promoter in the LacI producing pTrc99A cells.
The order of the concentration is used for more detailed assay with narrower range of the IPTG concentration.
Samples:
A4 placQI in pTrc99A
A4 ΔP in pTrc99A
Lux-lac hybrid promter + A4 in pBR322
Lux-lac hybrid promter + A4 in pTrc99A
Procedure:
prepare overnight culture for each sample
make fresh culture
take 3 ul of the overinight culture into 3 ml of LB (Amp and/or Kan) in Falcon tubes.
incubate for 2 to 3 hours until the observed OD is around 0.5
add AHL & IPTG solution
[AHL]final (in 3 ml LB culture) = 10 nM
[IPTG]final (in 3 ml LB culture) = 1000, 100, 10, 1, 0.1, 0.01, and 0 mM
incubate for 2 to 3 hours
apply 150 ul of samples into 96-well plaste
FLA measurement
