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

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(Autocreate 2013/07/18 Entry for Haynes_Lab:Notebook/Characterizing_AHL_quorum_sensing_homologs)
Current revision (00:49, 19 July 2013) (view source)
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==mm/dd/yyyy==
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==07/18/2013==
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* Insert content here...
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oligo annealing
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Mix oligos with annealing buffer
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<br>
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{| {{table}}
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|-
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|thing||rxn 1||rxn 2
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|-
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|O-006|| 1ul||3ul
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|-
 +
|O-008||1ul||3ul
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|-
 +
|O-011|| 1ul||3ul
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|-
 +
|O-012||1ul||3ul
 +
|-
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|O-013||1ul||3ul
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|-
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|buffer||2ul||2ul
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|-
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|H2O||13ul||3ul
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|-
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| || 20ul||20ul
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|}
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<br>
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boil 900ml water on hot plate until it's over 100deg C
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incubate tubes in boiling water for 10min
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cover in foil
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after 10min, turn off heat
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let cool for 6.5hours
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<br>
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cut 25ul J06702 with S/P in 30ul rxn
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ran on gel, gel ext
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<br>
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picked a bunch of sender and receiver colonies
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<br>
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cut oligos with X/P in 30ul rxn
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10 minutes 37
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added 1ul of phosphotase to dephosphorylate,
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10 minutes 37
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2mnutes 75
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ligation
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0.5 insert
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5ul vector
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5ul Roche 2x buffer
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1ul NEB T4
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left for 1:07 on the bench
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added 30ul middle bag DH5alphaT
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incubate on ice for 2 mins
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heat shock at 42 for 30s
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ice for 2 minutes
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plate
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 +
also transformed I13522 from EL plate in 30ul cells
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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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