User:Karmella Haynes/Notebook/BioBrick cloning/2009/11/18

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11/18/09 ✓ Cultures: KAH77(2), 78(3), 79(3), 83(2), 84(2), 85(2) ✓ Assemblies: KAH86, 87, 88, 89, 90, 91, 92 ✓ Oligo annealing: NLS myc stop (KAH011), K-stop (KAH012), BglII adapters Assemblies KAH86: HSVtkTATA-K (KAH48)/(S/P)/~3200 ✓ + (1) 2xYFP-NLSstop2-polyA-2xHS4 (KAH81)/(X/P)/2640 KAH87: (2) huPcCD/(E/S)/186+3200 + (8) mCh-VP64 (KAH74)/(E/X)/4061 KAH88: (3) mPcCD/(E/S)/186+3200 + " KAH89: (4) flyPcCD/(E/S)/186+3200 + " KAH90: (5) fshPcCD/(E/S)/186+3200 + " KAH91: (6) hCBX8/(E/S)/1170 + " KAH92: (7) flyPc/(E/S)/1173 + " > Digests (Fermentas FD) --> Gel purify 1, 6, 7, 8 --> Zymo clean & conc. 2-5 (for dephos.) Reagent Volume 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. KAH81 (X/P) 0.519 1.67 25.9 2. huPcCD (E/S) 1.590 1.76 79.5 3. mPcCD (E/S) 1.604 1.76 79.5 4. flyPcCD (E/S) 0.903 1.66 45.2 5. fshPcCD (E/S) 1.482 1.74 74.1 6. hCBX8 (E/S) 0.691 1.74 34.5 7. flyPc (E/S) 0.118 1.44 5.9 2. KAH74 (E/X) 0.188 1.37 9.4 > Dephosphorylation (Roche) Reagent Volume DNA (clean digest) up to 17 μL 10x buffer d.p. 2.0 phophatase 1.0 dH2O ---   20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL > Ligations (*Note: use insert + old backbone in calculations) Ligation Plate results (lig : neg crtl) 11/19/09 1. KAH81(X/P)/2640, 33 ng + KAH48(S/P)/~3200, 20 ng KAH86 >10:1 (Pick 2) 2. KAH48(S/P)/ 20 ng   3. huPcCD(E/S)/3386*, > 42 ng + KAH74(E/X)/4061, 25 ng KAH87 >10:1 (Pick 2) 4. mPcCD(E/S)/3386*, > 42 ng + " KAH88 >10:1 (Pick 2) 5. flyPcCD(E/S)/3386*, > 42 ng + " KAH89 >10:1 (Pick 2) 6. fshPcCD(E/S)/3386*, > 42 ng + " KAH90 >10:1 (Pick 2) 7. hCBX8(E/S)/1170, > 42 ng + " KAH91 >10:1 (Pick 2) 8. hCBX8(E/S)/1170, > 42 ng + " KAH92 >10:1 (Pick 2) 9. KAH74(S/P)/ 20 ng     1 2 3 4 5 6 7 8 9 Insert DNA 1.4 --- 1.7 1.7 1.7 1.7 0.5 2.5 --- Vector DNA 0.6 0.6 2.5 2.5 2.5 2.5 2.5 2.5 --- 2x lgn buf (Roche) 5.0 5.0 5.5 5.5 5.5 5.5 5.5 5.5 5.5 T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 dH2O 2.0 3.4 0.3 0.3 0.3 0.3 1.5 --- 2.0   10 μL 10 μL 11 μL 11 μL 11 μL 11 μL 11 μL 11.5 μL 11 μL Oligo annealing NLS myc stop (KAH011) K-stop (KAH012) Adapter BglII/EcoRI Adapter PstI/BglII DNA (oligos, 100 μM) up to 18 μL 10x annealing buffer 2.0 dH2O ---   20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight