Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2016/05/11

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05/11/2016

Yesterday, I picked 10 colonies from the pLas [1/2 plate]. Today I miniprepped 8 of them. Two out of the 10 liquid cultures did not grow. (Media was clear and no cells formed at the bottom of the tubes after I centrifuged them). Concentrations were in the 300ng/uL range (except for LasR 9—17ng/uL).

Next step is to digest verify plasmids with EcoRI and KpnI.

LasR 1 LasR 2 LasR 3 LasR 4 LasR 6 LasR 7 LasR 8 LasR 9
DNA 1.9 1.8 1.9 1.7 1.6 1.5 1.5 8
EcoRI 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
KpnI 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
10x FD 1 1 1 1 1 1 1 1
H20 6.1 6.2 6.1 6.3 6.4 6.5 6.5 0
Concentrations were similiar enough so I created a mastermix for plasmids 1-7 (skipping LasR 9 because of its low concentration)
Single x7
DNA 1.7 11.9
EcoRI 0.5 3.5
KpnI 0.5 3.5
10x FD 1 7
H20 6.3 44.1
Total 10 70

Next, I ran a gel with these digests. Results pending.