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

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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.
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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
1pTet-mCh 2/14/14 74ngP127P097533148
2LuxI sender const 8/31/13 179ngP063P06423654
3K084007 3/10/13 4-2-1P069P07032645
4K084007 3/10/13 4-2-2P069P07032645
5RBS-LasI 3/8/13 31ngP069P07032645


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 senderpTet-mCh 2/14/14 74ngP127P097533148



Ran on gel, only 5, LasI worked.

Ran a third, annealing temp 30

Label Template F Primer R Primer Annealing T Expected length
LuxILuxI sender const 8/31/13 179ngP063P06423654


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.


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