User:Karmella Haynes/Notebook/BioBrick cloning/2010/03/26

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03/26/10 ✓ Minipreps: KAH144 (4) ✓ Assembly: KAH145 Minipreps > Check with E/P digests Reagent Volume Expected: 1-4. KAH144 = 2399 15 μL/lane; 1% agarose DNA(plasmid) 2.0 μL 10X buffer 1.5 EcoRI 1.0 PstI 1.0 dH2O 9.5   15 μL --> 37°C/ ~15 min. Assemblies KAH145: (1) KAH144/(S/P)/~5599 + KAH51/(X/P)/1160 ✓ > Digests (Fermentas FD) Reagent Volume   30 μL/lane, 1% agarose DNA (plasmid) 10.0 10x buffer 3.0 enzyme 1 1.0 enzyme 2 1.0 dH2O 15.0   30 μL --> 37°C/ ~30 min. > Measure conc.'s Sample OD260 260/280 ng/μL 1. KAH144 (S/P) 0.217 1.91 10.8 --> KAH51 (X/P) = 42.9 > Ligations Ligation Plate results (lig : neg crtl) 03/28/10 1. KAH51(X/P)/1160, 10 ng + KAH144(S/P)/~5599, 25 ng KAH145 >10:1 (Pick 4) 2. KAH144(S/P)/ 25 ng ~ 20 colonies   1 2 Insert DNA 0.5 --- Vector DNA 2.5 2.5 2x lgn buf (Roche) 5.0 5.0 T4 ligase (NEB) 1.0 1.0 dH2O 1.0 1.5   10 μL 10 μL --> R.T./ ~10 min.; Add 50 μL Z-DH5α