Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2013/07/18

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07/18/2013

oligo annealing Mix oligos with annealing buffer

thingrxn 1rxn 2
O-006 1ul3ul
O-0081ul3ul
O-011 1ul3ul
O-0121ul3ul
O-0131ul3ul
buffer2ul2ul
H2O13ul3ul
20ul20ul


boil 900ml water on hot plate until it's over 100deg C incubate tubes in boiling water for 10min cover in foil after 10min, turn off heat let cool for 6.5hours
cut 25ul J06702 with S/P in 30ul rxn ran on gel, gel ext
picked a bunch of sender and receiver colonies
cut oligos with X/P in 30ul rxn 10 minutes 37 added 1ul of phosphotase to dephosphorylate, 10 minutes 37 2mnutes 75

ligation 0.5 insert 5ul vector 5ul Roche 2x buffer 1ul NEB T4

left for 1:07 on the bench

added 30ul middle bag DH5alphaT incubate on ice for 2 mins heat shock at 42 for 30s ice for 2 minutes plate

also transformed I13522 from EL plate in 30ul cells


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