User:Karmella Haynes/Notebook/BioBrick cloning/2011/08/01

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08/01/11 ✓ Assemblies: KAH231, 232, 233; KAH213, 219/MV1; KAH219, 223, 224/pSB31 blunt Assemblies KAH231: (1) Kozak/(S/P)/18 + KAH226/(X/P)/1564 ✓ KAH232: " + (2) KAH229/(X/P)/1756 KAH233: " + (3) KAH230/(X/P)/808 KAH213/MV1: (4) KAH213/(E/S)/2834 + MV1/(E/X)/5428 ✓ KAH219/MV1: (5) KAH219/(E/S)/2422 + " KAH219/pSB31: KAH219/(E/S blunt)/2422 ✓ + pSB31/(E blunt dp)/5127 ✓ KAH223/pSB31: KAH223/(E/S blunt)/1537 ✓ + " KAH224/pSB31: KAH224/(E/S blunt)/1837 ✓ + " > 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. Kozak (S/P) 0.461 2.04 23.1 2. KAH229 (X/P) 0.417 1.91 20.9 3. KAH230 (X/P) 0.115 2.53 5.8 4. KAH213 (E/S) 0.132 1.59 6.6 5. KAH219 (E/S) 0.158 2.07 7.9 > Ligations Ligation Plate results (lig : neg crtl) 08/02/11 1. KAH226(X/P)/1564, 19 ng + Kozak(S/P)/~3218, 20 ng KAH231 >10:1 (Pick 2) 2. KAH229(X/P)/1756, 22 ng + " KAH232 >10:1 (Pick 2) 3. KAH230(X/P)/808, 10 ng + " KAH233 >10:1 (Pick 2) 4. Kozak(S/P)/ 20 ng   5. KAH213(E/S)/2834, 21 ng + MV1(E/X)/5428, 20 ng KAH213/MV1 5:1 (Pick 3) 6. KAH219(E/S)/2422, 18 ng + " KAH219/MV1 5:1 (Pick 3) 7. MV1(E/X), 20 ng   8. KAH219(E/S bl)/2422, 19 ng + pSB31(E bl dp)/5127, 20 ng KAH219/pSB31 3:1 (Pick 4) 9. KAH223(E/S bl)/1537, 12 ng + " KAH223/pSB31 3:1 (Pick 4) 10. KAH224(E/S bl)/1837, 14 ng + " KAH224/pSB31 4:1 (Pick 4) 11. pSB31(E bl dp)/ 20 ng     1 2 3 4 5 6 7 8 9 10 11 Insert DNA 0.5 1.5 2.0 --- 3.2 2.2 --- 2.0 1.5 1.0 --- Vector DNA 1.0 1.0 1.0 1.0 0.5 0.5 0.5 0.8 0.8 0.8 0.8 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 2.5 1.5 1.0 3.0 0.3 1.3 3.5 1.2 1.7 2.2 3.2   10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL