Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2013/07/18: Difference between revisions
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Rene M Davis (talk | contribs) (Autocreate 2013/07/18 Entry for Haynes_Lab:Notebook/Characterizing_AHL_quorum_sensing_homologs) |
Rene M Davis (talk | contribs) |
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== | ==07/18/2013== | ||
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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07/18/2013oligo annealing
Mix oligos with annealing buffer
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 |