User:Karmella Haynes/Notebook/BioBrick cloning/2011/05/25

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05/25/11 ✓ Cultures/minipreps/digests: KAH184(12) ✓ Assemblies: KAH176fwd/183, 176fwd/184, 177fwd/183, 177fwd/184 Minipreps > Check with E/P digests Reagent Volume Expected: 1-12. KAH184 = 2191 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 KAH176fwd/183: (1) KAH183/(E/S)/2383 + (3) KAH176/MV8 /(E/X)/7785 KAH176fwd/184: (2) KAH184/(E/S)/2191 + " KAH177fwd/183: 5' part/(a/b)/2383 + (4) KAH177/MV8 /(E/X)/7978 KAH177fwd/184: 5' part/(a/b)/2191 + " > 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. KAH183 (E/S) 0.258 2.21 12.9 2. KAH184 (E/S) 0.225 1.84 11.2 3. KAH176 (E/X) 0.225 2.20 11.2 4. KAH177 (E/X) 0.393 2.12 19.7 > Ligations Ligation Plate results (lig : neg crtl) mm/dd/yy 1. KAH183(E/S)/2383, 18 ng + KAH176(E/X)/7785, 30 ng KAH183/176 2:1 (Pick 6) 2. KAH184(E/S)/2191, 16 ng + " KAH184/176 2:1 (Pick 6) 3. KAH176(E/X)/ 30 ng   4. KAH183(E/S)/2383, 18 ng + KAH177(E/X)/7978, 30 ng KAH183/177 3:1 (Pick 6) 5. KAH184(E/S)/2191, 16 ng + " KAH184/177 3:1 (Pick 6) 6. KAH177(E/X)/ 30 ng     1 2 3 4 5 6 Insert DNA 1.5 1.5 --- 2.5 2.5 --- Vector DNA 2.5 2.5 2.5 1.5 1.5 1.5 2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0 T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 dH2O --- --- 1.5 --- --- 2.5   10 μL 10 μL 10 μL 10 μL 10 μL 10 μL --> Add 10 μL to 30 μL DH5α-Turbo