User:Karmella Haynes/Notebook/BioBrick cloning/2010/07/25

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07/25/10

 * &#x2713; Assemblies: microMCS sites into FUW-Oct4 and pAct5C
 * &#x2713; Miniprep digests: check mut. mCh clones from last week

Digests > Mut mCh clones from last week > Check with BamHI/KpnI/PstI digests (plasmid = pJ10:G04381)

--> Send all 4 to Genewiz for sequencing on Monday

Assemblies
 * 1) KFV1: (1) pAct5C/(S/N)/~7000 + microMCS/(ΔN/ΔS dsOligo)/~35
 * 2) KAH015: (2) TetO-FUW-Oct4/(E)/8380 + microMCS2/(E/ΔE dsOligo)/~20

>Kinase rxn. and Oligo annealing --> Forgot to order microMCS2 oligos with 5' phosphates; re-do annealing rxn.

> Digests (Fermentas FD) --> FUW-Oct4 EcoRI fragment = 1316 --> pAct5C SpeI/NotI fragment (R. luciferase) = ~1000

> Measure conc.'s

> Dephosphorylation (Roche) --> TetO-FUW-Oct4/(E)

> Ligations

--> Re-do pAct5C/microMCS cloning with dp vector


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