IGEM:IMPERIAL/2006/LabCalendar/2006-9-7: Difference between revisions
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*Replaced supernatant of J37016 with saved supernatant from above | *Replaced supernatant of J37016 with saved supernatant from above | ||
*Put into shaker for 4hrs shake at 16:00 | *Put into shaker for 4hrs shake at 16:00 | ||
==Acylase Check== | |||
This was done to check whether acylase degrades anything that |
Revision as of 09:36, 7 September 2006
TO DO: *Electroporate Dr. Fray's plasmid containing aiiA into DH5a If any colonies grow until evening - culture up for testing on Friday !! *Maxi & mini of J37022, aiiA (with flag tag), aiiA *Next ligation step for new predator cell (with the new aiiA) once maxiprep is finished & electroporation on same day ! *Testing of J37015, J37022, Acylace Activity (need J37016 for two of them) *Send J37022 for sequencing *Make frozen stock of new T9002, J37036, J37022 !!! *Control digest for J37036 - Has ligation worked?
*What about the aiiA activity from Dr Fray's bacteria ? Have we cultured it ? Can we expect aiiA expression from the plasmid ?
*JS: Also, you guys need to start sending in parts to the registry, or else we won't have anything to show for all our efforts this summer!
Electroporation of Dr. Fray's plasmid containing aiiA
- Was electroporated this morning into Rosetta (not into DH5a because no expression will take place in DH5a due to the promoter not working in this strain)
- Might get some colonies so that it can be cultured for Western Blot tomorrow
Testing J37015
- Testing was carried out partly according to the current J37015 protocol and partly with some added measurements and amended steps. These are outlined below. (The protocol will be adjusted accordingly)
- Put into 2hrs shake at 9:45
- Taken out of shaker at 12:00
- Measure OD: 1.099 and dilute into 25mL to OD 0.1
- Spin down the cells & discard supernatant
- Add 25mL of prewarmed LB & resuspend
- Made up 2 x 25mL cultures
- One is inoculated with 1nM AHL (add 5μL of 5uM stock to the 25 mL)
- The other one is not inoculated with AHL
In Dr. Mann's Lab:
- Place in shaker
- Two samples were taken every 10-15min. for 2hrs
- One sample: Used Wallac-Victor III after taking samples to read fluorescence and OD (in order to detect GFP levels)
- Second sample: Supernatant is stored in order to assess AHL levels with J37016 later
Assessing AHL of samples with J37016:
- Replaced supernatant of J37016 with saved supernatant from above
- Put into shaker for 4hrs shake at 16:00
Acylase Check
This was done to check whether acylase degrades anything that