User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/04

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07/04/11
-->Note: incubate plates at 37C for 3 hours, put on bench overnight
 * Assemblies 1: KAH201, 202, 203 &#x2713;
 * Assemblies 2: Redo miR sensor assemblies (sub-set #1) (on hold: order more ligase)
 * Re-transformation: 5xGal4 KMC01 &#x2713;

Assemblies 1
 * 1) KAH201: 5xGal4 KMC01/(S/P)/93 &#x2713; + (1) KAH199/(X/P dp)/127
 * 2) KAH202: " + (2) KAH199/(X/P dp)/127
 * 3) KAH203: (3) 5xGal4 KMC01/(E/S dp)/93 + HSVtk TATA DB81/(X/P)/56 &#x2713;

> Digests (Fermentas FD) --> Specific notes

> Zymo clean --> Elute with 25 μL dH2O

> Measure conc.'s

> Dephosphorylation (Roche)

> Ligations

--> Add rxn. to 30 μL DH5α Turbo cells

Assemblies 2 --> Sequencing failed twice for miR sensor assemblies. Daniel tried ApaLI digest on a couple and saw no cutting (in previous digests, EcoRI probably cut the "vector"). Plasmids are strange. Re-do ligations, but check with ApaLI only from now on. --> Re-do miR sensor set 1, see 6/06/11

> Ligations

--> Note: Dilute vector to 20 ng/μL before use --> Use 2:1 ratio of insert, do not max out volume

--> Add rxn. to 25 μL DH5α Turbo cells


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