User:Karmella Haynes/Notebook/BioBrick cloning/2010/05/26

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05/26/10

 * &#x2713; MV3 and MV8 vector construction: transformation (C2925 dam-/dcm-)
 * &#x2713; MV3 and MV8 vectors construction: PCR hygromycin resistance from V0200

Transformation > Transform plasmids into C2925 dam-/dcm- cells (50 μL per transf.) > Use "quick and dirty" method (heat shock on plate)
 * 1) pcDNA3.1+ neo (MV1), 0.5 μL + 10 μL dH2O
 * 2) pcDNA3.1+ neo ΔpCMV (MV7), 0.5 μL + 10 μL dH2O
 * 3) dH2O, 10 μL

> 5/27/10: Lots of colonies on plasmid plates, zero on neg. ctrl. Success! 2 miniprep cultres per plasmid.

PCR > Amplify hygromycin resistance gene (1023 bp) from V0200 > Primers:
 * Hygro f1 (contains BsaBI site and start codon)
 * Hygro r1 (contains BstBI site)

--> BioRad Block A --> Zymo clean the PCR; elute w/ 20 μL dH2O (continue on 5/27)
 * 95°C/ 3 min.
 * [95°C/ 1 min., 57°C/ 1 min., 72°C/ 1 min.] x30
 * 72°C/ 3 min.
 * 4°C/ ∞

--> 60°C (BsaBI)/ 1 hr.; 65°C (BstBI)/ 1 hr. --> Gel purify

> Measure conc.


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