Team:Berkeley Wetlab/Passenger: MGFP

(Difference between revisions)

Revision as of 22:11, 18 October 2009

This assay tests for the presence of mgfp-5 displayed on the surface of E. coli through its ability to bind to the bottom of polystyrene plates.

Constructs used:

Experiment run with 5x replicate of each condition

Pick cells, using pin tool, with appropriate constructs from frozen stock plates into 96-well blocks with 1 ml of LB+AC media
Place blocks in 37C shaker overnight to grow to saturation

Set up 96-well blocks with 1 ml of LB+AC+arabinose media:
- Add 1 ul of arabinose at a concentration of (100mg/ml) to one ml of LB+AC media (1: 1000)
Take plates with constructs out of the 37C incubator and add 10 ul of each construct to the LB+AC+arabinose media to induce
Incubate blocks at 37C in shaker overnight.

Remove blocks from incubator
Pellet cells at 5400 rpm for 7 minutes
Remove supernatant by flicking block over a sink (swiftly inverting block in one motion)
Re-suspend cells in 100 ul of artificial seawater
Transfer 50 ul of cell suspensions with multi-pipette to labeled flat bottom 96-well costar plates (polystyrene plates)
Take OD 600 readings in TECAN
Cover plates with plastic film and let cells settle overnight

Uncover plates and flick out the liquid from each plate over a sink.
Wash each plate with 100 ul of artificial seawater (using dispense 100 ul program on the washing robot)
Flick out liquid again (this should remove the cells that are unbound to the surface of the polystyrene plates)
Add 25 ul of Tris buffer + 2% SDS to each well, to lyse cells
Set up BCA standards in empty wells on the plates for each condition replicate:
- 0.25, 0.75 and 1.0 mg of bovine serum albumin(BSA)/ ml of Tris Buffer + 2% SDS
Incubate plates at 50C for 15 minutes
While plates are incubating, set up another set of flat-bottom polystyrene plates with 190 ul of BCA reagents for each construct: BCA reagents are (1:50 ratio of BCA reagent (A) little blue bottle : (B) big white bottle.
Remove plates from incubator and transfer 10 ul of each lysate to the corresponding well in the plates with the BCA reagents.
Incubate plates for 40 minutes at 37C
Take OD 562 measurements on the TECAN
Analyze Data

@@ Line 83: / Line 83: @@
 [[Image:MGFP-nolink 9-30.jpg|900px]]
-==== Displayer only data from September 30th ====
+==== Displayers only data from September 30th ====
+[[Image: Displayers only 9-30.jpg|900px]]
 ==== One-tailed t-test analysis ====