SEED/2008/Day 8: Difference between revisions

Revision as of 11:19, 13 April 2008

Discuss beta-gal assay and AHL-inducible lacZ
Take 20ul of the culture into 80ul of the permeabilization solution (directly in cuvettes)
Wait 30m@30C
Measure the OD600 of the original cultures while waiting
Add 600ul substrate solution to permeabilized cells (right before hour lunch)
Record time of addition for each sample

grow up 2ml cultures expressing F2620.lacZa (BBa_T9003) with IPTG and different amounts of AHL
- 0, 1E-4, 1E-5, 1E-6, 1E-7, 1E-8, 1E-9, 1E-10
grow up 2ml control cultures of cells with and w/o lacZa expression plasmid
Make solutions as specified in Beta-Galactosidase_Assay_(A_better_Miller)

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 == Morning ==
-* dilute cells to log-phase growth
+* Discuss beta-gal assay and AHL-inducible lacZ
-* different amounts of induction with IPTG
+* Take 20ul of the culture into 80ul of the permeabilization solution (directly in cuvettes)
-* test F2620 with different amount of AHL + IPTG
+* Wait 30m@30C
-* sequencing analysis
+* Measure the OD600 of the original cultures while waiting
-** anything that's incorrect: pick another blue/green colony per plate for growth and templiphi in strips
+* Add 600ul substrate solution to permeabilized cells (right before hour lunch)
+* Record time of addition for each sample
 == Afternoon ==
-* beta-gal assay
+* As soon as come back from lunch, measure all samples
-* glycerol/set up sequencing as before for additional colonies
+* Every 20 minutes, make another measurement and record time
-* streak new plates of correct colonies to be tested
+* Calculate "Miller units"
+* Plot results
+* Compare results with existing F2620 data
 == Instructor Pre-prep ==
-* grow up cultures of Top10 w/ and w/o a lacZ-alpha expressing plasmid
+* grow up 2ml cultures expressing F2620.lacZa (BBa_T9003) with IPTG and different amounts of AHL
-* grow up cells expressing F2620.lacZa
+** 0, 1E-4, 1E-5, 1E-6, 1E-7, 1E-8, 1E-9, 1E-10
+* grow up 2ml control cultures of cells with and w/o lacZa expression plasmid
-== Instructor Post-prep ==
+* Make solutions as specified in [[Beta-Galactosidase_Assay_(A_better_Miller)]]
-* take out plates