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

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(Autocreate 2013/07/18 Entry for Haynes_Lab:Notebook/Characterizing_AHL_quorum_sensing_homologs)
 
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==mm/dd/yyyy==
==07/18/2013==
* Insert content here...
oligo annealing
Mix oligos with annealing buffer
<br>
{| {{table}}
|-
|thing||rxn 1||rxn 2
|-
|O-006|| 1ul||3ul
|-
|O-008||1ul||3ul
|-
|O-011|| 1ul||3ul
|-
|O-012||1ul||3ul
|-
|O-013||1ul||3ul
|-
|buffer||2ul||2ul
|-
|H2O||13ul||3ul
|-
| || 20ul||20ul
|}
<br>
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
<br>
cut 25ul J06702 with S/P in 30ul rxn
ran on gel, gel ext
<br>
picked a bunch of sender and receiver colonies
<br>
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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Revision as of 21:49, 18 July 2013

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07/18/2013

oligo annealing Mix oligos with annealing buffer

thing rxn 1 rxn 2
O-006 1ul 3ul
O-008 1ul 3ul
O-011 1ul 3ul
O-012 1ul 3ul
O-013 1ul 3ul
buffer 2ul 2ul
H2O 13ul 3ul
20ul 20ul


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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