User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/03

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12/03/09 ✓ Minipreps: KAH105(2), 106(2), 107(2), 108(2) ✓ Cultures: KAH93/MV2(2), 94/MV2(2), 95/MV2(2) ✓ Assemblies: KAH99, 100, 101, 105, 106, 107, 108 into MV2 Minipreps > Check with E/P/ScaI triple digests Reagent Volume Expected: 1,2. KAH105 = 2794 3,4. KAH106 = 2788 5,6. KAH107 = 2966 7,8. KAH108 = 2960 15 μL/lane; 1% agarose DNA(plasmid) 4.0 μL 10X buffer 1.5 EcoRI 1.0 PstI 1.0 ScaI 1.0 dH2O 6.5   15 μL --> 37°C/ ~15 min. Assemblies KAH99/MV2: (1) KAH99/(E/P)/2913 + MV2/(E/P)/4529 ✓ KAH100/MV2: (2) KAH100/(E/P)/3085 + " KAH101/MV2: (3) KAH101/(E/P)/3157 + " KAH105/MV2: (4) KAH105/(E/P)/2794 + " KAH106/MV2: (5) KAH106/(E/P)/2788 + " KAH107/MV2: (6) KAH107/(E/P)/2966 + " KAH108/MV2: (7) KAH108/(E/P)/2960 + " > Digests (Fermentas FD) --> E/P/ApaLI for KAH99-101 --> E/P/ScaI for KAH105-108 Reagent Volume   30 μL/lane, 1% agarose DNA (plasmid) up to 24 μL 10x buffer 3.0 EcoRI 1.0 PstI 1.0 enzyme 3 1.0 dH2O ---   30 μL --> 37°C/ ~30 min. > Measure conc.'s Sample OD260 260/280 ng/μL 1. KAH99(E/P) 0.405 1.99 20.27 2. KAH100(E/P) 0.429 1.93 21.43 3. KAH101(E/P) 0.398 2.09 19.90 4. KAH105(E/P) 0.198 1.92 9.88 5. KAH106(E/P) 0.240 2.18 11.98 6. KAH107(E/P) 0.225 1.99 11.23 7. KAH108(E/P) 0.221 2.58 11.06 > Ligations Ligation Plate results (lig : neg crtl) mm/dd/yy 1. KAH99(E/P)/2913, 32 ng + MV2(E/P)/4529, 25 ng KAH99/MV2 10:1 (Pick 2) 2. KAH100(E/P)/3085, 34 ng + " KAH100/MV2 10:1 (Pick 2) 3. KAH101(E/P)/3157, 35 ng + " KAH101/MV2 10:1 (Pick 2) 4. KAH105(E/P)/2794, 31 ng + " KAH105/MV2 10:1 (Pick 2) 5. KAH106(E/P)/2788, 31 ng + " KAH106/MV2 10:1 (Pick 2) 6. KAH107(E/P)/2966, 33 ng + " KAH107/MV2 10:1 (Pick 2) 7. KAH108(E/P)/2960, 33 ng + " KAH108/MV2 10:1 (Pick 2) 8. MV2(E/P)/ 25 ng     1 2 3 4 5 6 7 8 Insert DNA 1.6 1.6 1.8 3.0 2.6 3.0 3.0 --- Vector DNA 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 2x lgn buf (Roche) 5.0 5.0 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 1.0 1.0 dH2O 1.4 1.4 1.2 --- 0.4 --- --- 3.0   10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL --> R.T./ 10 min.; Add 25 μL Z-DH5α