User:Karmella Haynes/Notebook/BioBrick cloning/2010/04/09

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04/09/10 ✓ Minipreps: KAH142 rebuilt (4) ✓ Assemblies: KAH146, 147 (H3me3 reporters) and KAH148-153 (Pc-ATF's w/o VP64, controls) Minipreps > Check with E/P digests Reagent Volume Expected: 1-4. KAH142 = 4571 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 KAH146: (1) KAH96/(E/S/ApaL1)/2825 + (3) KAH145/(E/X)/~6755 KAH147: (2) KAH142/(E/S)/4571 + " KAH148: KAH01/(E/S dp)/186 + (4) KAH116/(E/X)/~3975 KAH149: KAH02/(E/S dp)/186 + " KAH150: KAH03/(E/S dp)/186 + " KAH151: KAH04/(E/S dp)/186 + " KAH152: KAH06/(E/S)/1170 + " KAH153: KAH07/(E/S)/1173 + " > Digests (Fermentas FD) --> Triple digest KAH96 w/ E/S/ApaLI Reagent Volume   30 μL/lane, 1% agarose DNA (plasmid) up to 25 μL up to 24 μL 10x buffer 3.0 3.0 enzyme 1 1.0 1.0 enzyme 2 1.0 1.0 ApaLI --- 1.0 dH2O --- ---   30 μL 30 μL --> 37°C/ ~30 min. > Measure conc.'s Sample OD260 260/280 ng/μL 1. KAH96 (E/S/ApaLI) 0.393 1.75 19.6 2. KAH142 (E/S) 0.394 1.91 19.7 3. KAH145 (E/X) 0.371 1.71 18.6 4. KAH116 (E/X) 0.762 1.85 38.1 > Ligations Ligation Plate results (lig : neg crtl) 04/10/10 1. KAH96(E/S)/2825, 33 ng + KAH145(E/X)/6755, 40 ng KAH146 1:1 (re-do) 2. KAH142(E/S)/4571, 54 ng + " KAH147 1:1 (re-do) 3. KAH145(E/X)/ 40 ng ~50 colonies 4. KAH01(E/S)/186, 2 ng + KAH116(E/X)/3975, 20 ng KAH148 1:1 (re-do) 5. KAH02(E/S)/186, 2 ng + " KAH149 1:1 (re-do) 6. KAH03(E/S)/186, 2 ng + " KAH150 1:1 (re-do) 7. KAH04(E/S)/186, 2 ng + " KAH151 1:1 (re-do) 8. KAH06(E/S)/1170, 12 ng + " KAH152 1:1 (re-do) 9. KAH06(E/S)/1173, 12 ng + " KAH153 1:1 (re-do) 10. KAH116(E/X)/ 50 ng ~300 colonies   1 2 3 4 5 6 7 8 9 10 Insert DNA 1.8 2.8 --- 0.5 0.5 0.5 0.5 0.5 0.5 --- Vector DNA 2.2 2.2 2.2 0.5 0.5 0.5 0.5 0.5 0.5 0.5 2x lgn buf (Roche) 6.0 6.0 6.0 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 1.0 1.0 dH2O 1.0 --- 2.8 3.0 3.0 3.0 3.0 3.0 3.0 3.5   12 μL 12 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL --> R.T./ ~10 min.; Add to 20 μL Z-DH5α