User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/14

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12/14/09 ✓ Minipreps: digests for 12/12/09 minipreps ✓ Cultures: KAH123(3), 124(3), 125(3) ✓ Assemblies: KAH126-133 into MV2 Minipreps > Check with E/P digests Reagent Volume Expected: 1,2. KAH126 = 1880 3,4. KAH127 = 1880 5,6. KAH128 = 1880 7,8. KAH129 = 1880 9,10. KAH130 = 2864 11,12. KAH131 = 2867 13,14. KAH132 = 1688 15,16. KAH133 = 1532 17,18. KAH134 = 1400 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 KAH126/MV2: (1) KAH126/(E/P)/1880 + MV2/(E/P)/4529 ✓ KAH127/MV2: (2) KAH127/(E/P)/1880 + " KAH128/MV2: (3) KAH128/(E/P)/1880 + " KAH129/MV2: (4) KAH129/(E/P)/1880 + " KAH130/MV2: (5) KAH130/(E/P)/2864 + " KAH131/MV2: (6) KAH131/(E/P)/2867 + " KAH132/MV2: (7) KAH132/(E/P)/1688 + " KAH133/MV2: (8) KAH133/(E/P)/1532 + " > Digests (Fermentas FD) --> Triple digest KAH130 & 131 w/ E/P/ScaI Reagent Vol (1-4, 7, 8) Vol (5, 6) 30 μL/lane, 1% agarose DNA (plasmid) 15.0 15.0 10x buffer 3.0 3.0 enzyme 1 1.0 1.0 enzyme 2 1.0 1.0 ScaI --- 1.0 dH2O 10.0 9.0   30 μL 30 μL --> 37°C/ ~30 min. > Measure conc.'s Sample OD260 260/280 ng/μL 1. KAH126(E/P) 0.705 1.86 35.2 2. KAH127(E/P) 1.095 1.88 54.7 3. KAH128(E/P) 0.916 1.88 45.8 4. KAH129(E/P) 0.892 1.85 44.6 5. KAH130(E/P) 0.776 2.02 38.8 6. KAH131(E/P) 0.620 1.93 31.0 7. KAH132(E/P) 0.729 1.88 36.5 8. KAH133(E/P) 0.380 1.96 19.0 > Ligations Ligation Plate results (lig : neg crtl) 12/15/09 1. KAH126(E/P)/1880, 21 ng + MV2/(E/P)/4529, 25 ng KAH126/MV2 10:1 (Pick 2) 2. KAH127(E/P)/1880, 21 ng + " KAH127/MV2 10:1 (Pick 2) 3. KAH128(E/P)/1880, 21 ng + " KAH128/MV2 10:1 (Pick 2) 4. KAH129(E/P)/1880, 21 ng + " KAH129/MV2 10:1 (Pick 2) 5. KAH130(E/P)/2864, 32 ng + " KAH130/MV2 10:1 (Pick 2) 6. KAH131(E/P)/2867, 32 ng + " KAH131/MV2 10:1 (Pick 2) 7. KAH132(E/P)/1688, 19 ng + " KAH132/MV2 10:1 (Pick 2) 8. KAH133(E/P)/1532, 17 ng + " KAH133/MV2 10:1 (Pick 2) 2. MV2(E/P)/ 25 ng     1 2 3 4 5 6 7 8 9 Insert DNA 1.0 1.0 1.0 1.0 1.5 1.5 1.0 1.0 --- Vector DNA 1.0 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 5.0 T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 dH2O 2.0 2.0 2.0 2.0 1.5 1.5 2.0 2.0 3.0   10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL --> R.T./ ~10 min.; Add 20μL Z-DH5α