Tokyo/IPTG assay
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===Procedure: === | ===Procedure: === | ||
| - | [[Image:IPTGassay.JPG|thumb|200px| '''Fig.1: '''Exogenously added | + | [[Image:IPTGassay.JPG|thumb|200px| '''Fig.1: '''Exogenously added IPTG]] |
<br>prepare overnight culture for each sample | <br>prepare overnight culture for each sample | ||
<br>make fresh culture | <br>make fresh culture | ||
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===Result & Conclusion: === | ===Result & Conclusion: === | ||
| - | [[Image:hill IPTG.JPG|thumb|200px| '''Fig. | + | [[Image:hill IPTG.JPG|thumb|200px| '''Fig.2: '''IPTG assay graph]] |
Revision as of 05:22, 24 October 2007
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
Result & Conclusion:
