Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2014/05/12

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Ran PCR products from 5/6/14 on a gel. Only 6, RhlI worked.

Re-ran other PCRs, annealing temp 40, 4min ext

Number Template F Primer R Primer Annealing T Expected length
1 pTet-mCh 2/14/14 74ng P127 P097 53 3148
2 LuxI sender const 8/31/13 179ng P063 P064 23 654
3 K084007 3/10/13 4-2-1 P069 P070 32 645
4 K084007 3/10/13 4-2-2 P069 P070 32 645
5 RBS-LasI 3/8/13 31ng P069 P070 32 645


Ran a second one of the backbone with annealing temp 50, ext time 4 min

Label Template F Primer R Primer Annealing T Expected length
mCh sender pTet-mCh 2/14/14 74ng P127 P097 53 3148



Ran on gel, only 5, LasI worked.

Ran a third, annealing temp 30

Label Template F Primer R Primer Annealing T Expected length
LuxI LuxI sender const 8/31/13 179ng P063 P064 23 654


Realized P127 and P097 will never work on pTet-mCh because after the first round with one primer, the binding site for the other one is at the wrong end. Will need to redesign one of the primers to add a second RBS in front of mCherry or behind pTet.