Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2016/01/21

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01/21/2016

Create RhlI, RhlR, BjaI, and BjaR glyercol stocks

Label tubes with each plasmid's name
Add 500uL of glycerol diluted in 50% water into four 1.5mL tubes.
Pipet 500uL of each liquid culture into their respective tubes.
Mix
Store in a box labeled "Renè, Cass, Jiaqi, Tim glycerol stocks" in -80°C freezer.
For use:
Touch tip of pipette to the surface of glycerol stock
Dip pipette tip into liquid media and begin growing!
(This skips the transformation step!)

Try PCR for gBlock again

' Volume (uL)
DNA 1
10uM F Primer1
10uM R Primer1
10uM dNTPs1
Phusion0.5
5x HF Buffer10
DMSO (optional)
Water35.5
Total50

Begin RhlR and RhlI growth curve experiments!

In a 96-well plate, we pipetted 100uL of LB into well 1, 100uL of RhlI liquid culture into well 2, and 100uL of RhlR culture into well 3.
We placed the liquid cultures back in the incubator to shake.
Then, we used the program RenéRyanJiaqiOD in the plate reader to check the well's OD, GFP, and RFP.
We recorded this data and then placed the well in the incubator to shake and grow.
Every hour or half-hour, we repeated this process, adding LB, RhlI, and RhlR into the next three wells, and taking a reading of all the wells.
We began to notice that cells grow quicker (OD increases faster) in liquid media tubes compared to in the wells. This is something to take account of if we grow the cultures in a well instead of in a tube during the experiment.


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