User:Karmella Haynes/Notebook/BioBrick cloning/2009/11/27

Project name

Main project page Previous entry      Next entry

11/27/09 Assemblies: KAH99, 100, 101, 102, 103, 104 Assemblies KAH99: (1) Ubc-5xGal4 (KAH83)/(S/P)/~3643 + (4) K-PEST-2xAmCyan-NLS-stop-polyA(KAH82)/(X/P)/2464 KAH100: (2) HPK-5xGal4 (KAH84)/(S/P)/~3815 + " KAH101: (3) CMV-5xGal4 (KAH85)/(S/P)/~3887 + " KAH102: Ubc/(E/S)/344 ✓ + (5) Kstop(KAH012)/(E/X)/~3200 KAH103: HPK/(E/S)/516 ✓ + " KAH104: HPK/(E/S)/516 ✓ + K/(E/X)/~3200 ✓ > Digests (Fermentas FD) Reagent Volume   30 μL/lane, 1% agarose 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. KAH83 (S/P) 0.420 1.80 21.01 2. KAH84 (S/P) 0.213 1.62 10.67 3. KAH85 (S/P) 0.466 1.83 23.30 4. KAH82 (X/P) 0.645 1.82 32.24 5. KAH012 (E/X) 0.278 1.80 13.92 > Ligations Ligation Plate results (lig : neg crtl) 11/29/09 1. KAH82(X/P)/2464, >41 ng + KAH83(S/P)/3643, 30 ng KAH99 >10:1 (Pick 2) 2. KAH83(S/P)/ 30 ng   3. KAH82(X/P)/2464, >39 ng + KAH84(S/P)/3815, 30 ng KAH100 >10:1 (Pick 2) 4. KAH84(S/P)/ 30 ng   5. KAH82(X/P)/2464, >38 ng + KAH85(S/P)/3887, 30 ng KAH101 >10:1 (Pick 2) 6. KAH85(S/P)/ 30 ng   7. Ubc(E/S)/344, >4 ng + Kstop(E/X)/3200, 20 ng KAH102 >10:1 (Pick 2) 8. HPK(E/S)/516, >6 ng + " KAH103 >10:1 (Pick 2) 9. Kstop(E/X)/ 20 ng   10.HPK(E/S)/516, >6 ng + K(E/X)/3200, 20 ng KAH104 >10:1 (Pick 2) 11. K(E/X)/ 20 ng     1 2 3 4 5 6 7 8 9 10 11 Insert DNA 1.3 --- 1.2 --- 1.2 --- 0.4 0.6 --- 0.6 --- Vector DNA 1.4 1.4 2.8 2.8 1.3 1.3 1.4 1.4 1.4 0.6 0.6 2x lgn buf (Roche) 5.0 5.0 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 1.0 1.0 dH2O 1.3 2.6 --- 1.2 1.5 2.7 2.2 2.0 2.6 2.8 3.4   10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL 10μL ---> R.T./ ~10 min.; Add 18 μL Z-DH5α