Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2014/05/23

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mm/dd/yyyy Colony PCR on Sender Constructs F primer P093 R primer P101 RhlI - 10 colonies EsaI - 10 colonies LasI - 10 colonies + ctrl 1 rxn Positive control is pTet-mCherry 1:1000 miniprepped vector, which should be what a religated backbone is. 10μL reactions, annealing T 41°C, extension time 1:30 Picked each of the colonies on the receiver plates I made trying to test if any of the constructs were correct. I picked a big swab of each colony and swirled it in some PBS. Then I ran each of them on the flow cytometer. I used the senders (which definitely have GFP) as positive controls. The RhlI sender was green and every. other. colony. was not. This is clearly not a clever and efficient way to screen for receiver constructs. Possible reasons for failure: All of the receiver constructs are incorrect. Receivers are making very low amounts of GFP because of their medium RBS, reduction of read-through expression, and being in DH5alphas. Senders were not making much AHL because they're in DH5alphas. Maybe I didn't leave them long enough in the incubator.