User:Karmella Haynes/Notebook/BioBrick cloning/2010/04/14

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04/14/10

 * &#x2713; Minipreps: KAH96, 142, 146, 147/V0201 (12 total); KAH154-159 (12 total)
 * &#x2713; Assemblies: KAH154-159/MV2
 * &#x2713; Cultures: KAH142(6), 146(6), 147/V0201(8)
 * &#x2713; Plasmid transformations: MV2 and V0201

Minipreps > Check KAH96, 142, 146, 147/V0201 with XbaI digest (add 641 to expected insert size) > Check KAH154-159 with E/P digest

--> 37°C/ ~15 min.

--> Pick more colonies for KAH142, 146, 147/V0201

Assemblies
 * 1) KAH154/MV2: (1) KAH154/(E/P)/1724 + (7) MV2/(E/P)/4529
 * 2) KAH155/MV2: (2) KAH155/(E/P)/1724 + "
 * 3) KAH156/MV2: (3) KAH156/(E/P)/1724 + "
 * 4) KAH157/MV2: (4) KAH157/(E/P)/1724 + "
 * 5) KAH158/MV2: (5) KAH158/(E/P)/2688 + "
 * 6) KAH159/MV2: (6) KAH159/(E/P)/2691 + "

> Digests (Fermentas FD) --> Triple digest KAH158, 159 w/ E/P/ScaI --> Digest for 1 hour to ensure complete digestion

> Measure conc.'s

> Dephosphorylation (Roche)
 * 1) MV2 (E/P)

> Ligations

--> R.T./ ~10 min.; Add 28 μL Turbo DH5α (T-DH5α); ice/ 5 min. --> plate on warm LB, 100 μg/mL Amp; R.T. overnight; 37 °C next day for ~3 hours


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