User:Karmella Haynes/Notebook/BioBrick cloning/2011/05/20

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05/20/11 ✓ Assembly: Remove PEST from CFP reporter gene; KAH181 ✓ Retransform: KAH57 Assemblies KAH181: Kozak/(S/P)/~3200 ✓ + (1) KAH57/(X/P)/1426 > Digests (Fermentas FD) Reagent Volume   30 μL/lane, 1% agarose DNA (plasmid) up to 25 μL 10x buffer 3.0 enzyme 1 1.0 enzyme 2 1.0 dH2O ---   30 μL --> 37°C/ ~30 min. > Measure conc.'s Sample OD260 260/280 ng/μL 1. KAH57 (X/P) 0.543 1.81 27.2 > Ligations Ligation Plate results (lig : neg crtl) 05/21/11 1. KAH57(X/P)/1426, 22 ng + Kozak(S/P)/~3200, 25 ng KAH181 >10:1 (Pick 2) 2. Kozak(S/P)/ 25 ng     1 2 Insert DNA 1.0 --- Vector DNA 0.5 0.5 2x lgn buf (Roche) 5.0 5.0 T4 ligase (NEB) 1.0 1.0 dH2O 2.5 3.5   10 μL 10 μL --> Add 10 μL lgn rxn. to 30 μL Dh5α Turbo --> Also do retransformation of KAH57