Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2015/05/20

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05/20/2015

We mini-prepped receivers and synthases for gel verification.

Sample Read# 260 280 260/280 ng/µL
AubR 1 0.711 0.371 1.914 711.002
AubR 2 0.628 0.333 1.885 628.208
BraR 1 0.824 0.433 1.904 824.021
BraR 2 0.485 0.255 1.906 485.463
RpaR 1 0.705 0.369 1.91 705.115
RpaR 2 0.572 0.304 1.886 572.435
AubI 1 0.399 0.21 1.902 399.31
AubI 2 0.563 0.313 1.8 563.178
AubI 3 0.567 0.299 1.895 567.352
BjaI 1 0.577 0.304 1.898 577.174
BjaI 2 0.561 0.292 1.921 561.178
BjaI 3 0.589 0.312 1.891 589.038
Sample Read# 260 280 260/280 ng/µL
BraI 1 0.542 0.281 1.933 542.456
BraI 2 0.59 0.306 1.926 589.609
CerI 1 0.638 0.333 1.916 637.778
CerI 2 0.789 0.413 1.911 789.27
EsaI 1 0.573 0.298 1.922 572.722
EsaI 2 0.216 0.114 1.899 215.855
LasI 1 0.373 0.195 1.915 373.17
LasI 2 0.172 0.089 1.923 171.718
RpaI 1 0.406 0.214 1.899 405.978
RpaI 2 0.646 0.336 1.921 646.406
SinI 1 0.798 0.42 1.898 797.594
SinI 2 0.691 0.364 1.9 691.117

The synthases were double digested with EcoRI and XbaI; Receivers were digested with KpnI and EcoRI. Here is a gel of the digestion reactions:
File:20.05.15 synthase and receiver digest verification-cropped-edited.tiff
Based on the gel, we think the following colonies worked: BraI 2, CerI (1?), 2, EsaI 2, RpaI1 for the synthases. [ ] for the receivers. (all?). Ryan will send them in for sequencing to verify. Meanwhile, we will pick more colonies from the ones that didn't work and repeat the process.