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

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'''Colony PCR on Sender Constructs'''<br>
F primer P093<br>R primer P101<br>RhlI - 10 colonies<br>EsaI - 10 colonies<br>LasI - 10 colonies<br>+ ctrl 1 rxn<br>Positive control is pTet-mCherry 1:1000 miniprepped vector, which should be what a religated backbone is.<br>10μL reactions, annealing T 41°C, extension time 1:30
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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.  
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.  
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Revision as of 15:52, 29 May 2014

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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.



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