User:Karmella Haynes/Notebook/BioBrick cloning/2009/11/20

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11/20/09 Minipreps: KAH86(2), 87(2), 88(2), 89(2), 90(2), 91(2), 92(2); KAH84(2, re-pick) Digests: KAH77, 78, 79 (E/P/ScaI triple digests) Assemblies: KAH011, 012; KAH93, 94, 95 Minipreps > Check with E/P digests > Include KAH77-79 E/P/ScaI digests (ScaI cuts V0120) Reagent Volume   Expected: 1,2. KAH86 = 2726 3,4. KAH87 = 1053 5,6. KAH88 = 1053 7,8. KAH89 = 1053 9,10. KAH90 = 1053 11,12. KAH91 = 2037 13,14. KAH92 = 2040 15,16. KAH84 = 615 17,18. KAH77 = 3480 (& 1784, 1439) 19-21. KAH78 = 3652 (& 1784, 1439) 22-24. KAH79 = 3724 (& 1784, 1439) 15μL/lane, 1% gel   (1-16) (17-24) DNA(plasmid) 2.0 1.0 10X buffer 1.5 1.5 EcoRI 1.0 1.0 PstI 1.0 1.0 ScaI --- 1.0 dH2O 9.5 15 μL --> 37°C/ ~15 min. Assemblies KAH011: NLS-myc-stop/(X/P ds-oligo)/64 ✓ + (1) V0120/(X/P)/3200 KAH012: Kstop/(X/P ds-oligo)/24 ✓ + " KAH93: (2) 2xHS4-Ubc-K-Gal4DB-PEST-2xmCh-NLS-stop (KAH77)/(E/S)/3480 + (5) 5xLuc target-polyA-2xHS4 (KAH67)/(E/X)/4553 KAH94: (3) 2xHS4-HPK-K-Gal4DB-PEST-2xmCh-NLS-stop (KAH78)/(E/S)/3652 + " KAH95: (4) 2xHS4-CMV-K-Gal4DB-PEST-2xmCh-NLS-stop (KAH79)/(E/S)/3724 + " > Digests (Fermentas FD) --> Triple digest KAH977-79 to cut V0120 Reagent Volume   Isolate: 1. V0120 = 2726 2. KAH77 = 3480 3. KAH78 = 3652 4. KAH79 = 3724 5. KAH67 = 4553 30 μL/lane, 1% gel   (1,5) (2-4) DNA (plasmid) up to 25 μL 5 10x buffer 3.0 3.0 enzyme 1 1.0 1.0 enzyme 2 1.0 1.0 ScaI --- 1.0 dH2O --- 19   30 μL 30 μL --> 37°C/ ~30 min. > Measure conc.'s Sample OD260 260/280 ng/μL 1. V0120(X/P) 0.097 2.87 4.83 2. KAH77(E/S) 0.405 2.13 20.24 3. KAH78(E/S) 0.432 2.07 21.62 4. KAH79(E/S) 0.396 2.06 19.79 5. KAH67(E/X) 1.885 1.87 94.25 > Ligations Ligation Plate results (lig : neg crtl) mm/dd/yy 1. KAH011(X/P)/64, > 0.8 ng + V0120(X/P)/3200, 20 ng KAH011 10:1 (Pick 2) 2. KAH012(X/P)/24, > 0.3 ng + V0120(X/P)/3200, 20 ng KAH012 >10:1 (Pick 2) 3. V0120(X/P) 20 ng   4. KAH77(E/S)/3480, > 46 ng + KAH67(E/X)/4553, 30 ng KAH93 3:1 (Pick 2) 5. KAH78(E/S)/3652, > 48 ng + " KAH94 5:1 (Pick 2) 6. KAH79(E/S)/3724, > 49 ng + " KAH95 5:1 (Pick 2) 7. KAH67(E/X) 30 ng   1 2 3 4 5 6 7 Insert DNA 0.5 0.5 --- 2.5 2.5 2.5 --- Vector DNA 4.0 4.0 4.0 0.5 0.5 0.5 0.5 2x lgn buf (Roche) 5.5 5.5 5.5 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 dH2O --- --- 0.5 1.0 1.0 1.0 3.5   11 μL 11 μL 11 μL 10 μL 10 μL 10 μL 10 μL --> R.T./ 10 min.; Add 25 μL Z-DH5α