SEED/2008/Day 8: Difference between revisions
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* Discuss beta-gal assay and AHL-inducible lacZ | * Discuss beta-gal assay and AHL-inducible lacZ | ||
* Take 20ul of the culture into 80ul of the permeabilization solution (directly in cuvettes) | * Take 20ul of the culture into 80ul of the permeabilization solution (directly in cuvettes) | ||
* | * Leave at 30C over lunch | ||
* Measure the OD600 of the original cultures while waiting | * Measure the OD600 of the original cultures while waiting | ||
== Afternoon == | == Afternoon == | ||
* | * Add 600ul substrate solution to permeabilized cells | ||
* Every 20 minutes, make another measurement and record time | * Every 20 minutes, make another measurement and record time | ||
* | |||
== Homework == | |||
* Analyze data | |||
* Plot results | * Plot results | ||
* Compare results with existing F2620 data | * Compare results with existing F2620 data | ||
== Instructor Pre-prep == | == Instructor Pre-prep == | ||
* grow up | * grow up 1ml cultures expressing F2620.lacZa (BBa_T9003) with IPTG and different amounts of AHL | ||
** 0 | ** 0, 1E-5, 1E-6, 1E-7, 1E-8, 1E-9, 1E-10, 1E-11 | ||
* grow up | * grow up 1ml control cultures of cells with and w/o lacZa expression plasmid | ||
* Make solutions as specified in [[Beta-Galactosidase_Assay_(A_better_Miller)]] | * Make solutions as specified in [[Beta-Galactosidase_Assay_(A_better_Miller)]] | ||
Latest revision as of 09:19, 21 April 2008
Morning
- Discuss beta-gal assay and AHL-inducible lacZ
- Take 20ul of the culture into 80ul of the permeabilization solution (directly in cuvettes)
- Leave at 30C over lunch
- Measure the OD600 of the original cultures while waiting
Afternoon
- Add 600ul substrate solution to permeabilized cells
- Every 20 minutes, make another measurement and record time
Homework
- Analyze data
- Plot results
- Compare results with existing F2620 data
Instructor Pre-prep
- grow up 1ml cultures expressing F2620.lacZa (BBa_T9003) with IPTG and different amounts of AHL
- 0, 1E-5, 1E-6, 1E-7, 1E-8, 1E-9, 1E-10, 1E-11
- grow up 1ml control cultures of cells with and w/o lacZa expression plasmid
- Make solutions as specified in Beta-Galactosidase_Assay_(A_better_Miller)
