Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2014/04/30

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Purified PCR reactions 5 and 6 from yesterday (receiver backbone) using the gel extraction columns and the DNA clean and concentrator kit. Got really low yield, need to look into a better kit for this.

Will set up golden gate with the purified PCR backbones. Will gel extract the other ones and then run those tomorrow if this doesn't work. Will only do one of each because I don't have enough receiver backbone.

Description ng/µL length fmol/ul ul for 200 fmol
Rec bb 31 2203 21 9.52
pLux-GFP 47.148 1051 68 2.94
pRhl-GFP 50.466 1051 72 2.78
pEsa-GFP 32.1 1051 46 4.35
pLac-LuxR 45.771 817 89 2.25
pLac-RhlR 59.88 765 119 1.68
pLac-EsaR 37.847 758 74 2.70


Reaction Rec bb pLux-GFP pLac-LuxR BsmBI T4 ligase T4 ligase buffer H2O
Lux Rec 9.52 2.94 2.25 0.5 1.25 2 1.54
Rhl Rec 9.52 2.78 1.68 0.5 1.25 2 2.27
Esa Rec 9.52 4.35 2.7 0.5 1.25 2 -0.32
Bb control 9.52 0 0 0.5 1.25 2 6.73



Couldn't see the PCR products on the gel, maybe I ran it too long? 1 hour at 100, 1.5 more at 85. Half gel. Should have at least been able to see the the top of the 10kb ladder but I couldn't see anything. Wondering if the gel box UV light is bad.



Transformation of golden gate reactions. New cells made with Behzad. Used 70ul for each of 5 transformations. 30 minutes on ice, 30s at 42, 2 min ice, 1 hour shaking SOC.