Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2013/03/12

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Stopped the sender cultures at noon. Spun in 2ml tubes for 3 minutes at 13.1g. Poured supernatants into 15ml conical tubes. Added 5ml LB to the round bottom tunes. Started shaking again at 1:30pm.


Afternoon: Added 5ul of 50mM AHL to 5ml water, added 1ml of hexane to that, mixed vigorously, can also heat
Mess with ratio, can heat it also
Can centrifuge it down to help break it up
Move top layer directly to HPLC vial
Put vial in the right spot
Hit start


GC
Open all 3 cylinders
Check pressures after turning the machines on
Turn on top Hewlett Packard thing
Turn on GC machine (in back on right side)
Open software
Open fiD air A detector
Close hydrogen
Push the FiD igniter (instructions on wall for this part)
Check that it's lit by looking down the hole using the wrench as a mirror and see if condensation forms on it
If any data ever looks funny, check that condensation still forms on the wrench

Software

Sequence parameters
Signal 2 prefix
Make sure it goes to my directory
Method is AHLm

Flame ionization detector


Controls
5 step calibration curve
.5ml hexane
2ul
1.5ul
1ul
.5ul
.25ul


Started at 340, 23 samples



tonight:

miniprepped E0420 (E0343 didn't grow)

cut with X & P

cut K092900 with S & P

gel extracted

ligated


ligation:

3.8ul backbone

13ul insert

behzad's roche buffer and ligase

not doing backbone control



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