User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/19

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07/19/11 ✓ Cultures/minipreps: started cultures last night, KAH219, 221, 222, 223, 224 (4 each) ✓ Assemblies: KAH213, 214, 215, 219, 220, 221/MV1 ✓ Cultures: KAH219, 223, 224/pSB31 (8 each) (o/n) ✓ Cultures: KAH220, 221, 222/pSB31 (5 ml, 2 each) (o/n) Minipreps > Check with E/P digests Reagent Volume Expected: pSB31 = 4500, 450 *pSB31 bands **last frag + 300 1-4. 219/pSB31 5-8 = 4500*, 150*, 444, 1735, 532** 5-8. 221/pSB31 5-8 = 4500*, 150*, 444, 1490** 9-12. 222/pSB31 5-8 = 4500*, 150*, 636, 1735, 532** 13-16. 223/pSB31 5-8 = 4500*, 150*, 636, 1190** 17-20. 224/pSB31 5-8 = 4500*, 150*, 636, 1490** 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. --> 221/pSB31 and 222/pSB31 worked. Pick more colonies for others. Assemblies KAH213/MV1: (1) KAH213/(E/S)/2834 + MV1/(E/X)/5428 KAH214/MV1: (2) KAH214/(E/S)/1757 + " KAH215/MV1: (3) KAH215/(E/S)/2057 + " KAH219/MV1: KAH219/(E/S)/2422 + " KAH220/MV1: KAH220/(E/S)/1345 + " KAH221/MV1: KAH221/(E/S)/1757 + " KAH222/MV1: KAH222/(E/S)/2614 + " KAH223/MV1: KAH223/(E/S)/1537 + " KAH224/MV1: KAH224/(E/S)/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. KAH213 (E/S) 0.174 1.88 8.7 2. KAH214 (E/S) 0.227 1.84 11.3 3. KAH215 (E/S) 0.206 2.61 10.3 > Ligations Ligation Plate results (lig : neg crtl) mm/dd/yy 1. KAH213(E/S)/2834, 26 ng + MV1(E/X)/5428, 25 ng KAH213/MV1 #:1 (Pick #) 2. KAH214(E/S)/1757, 16 ng + " KAH214/MV1 #:1 (Pick #) 3. KAH215(E/S)/2057, 19 ng + " KAH215/MV1 #:1 (Pick #) 4. KAH219(E/S)/2422, 22 ng + " KAH219/MV1 #:1 (Pick #) 5. KAH220(E/S)/1345, 12 ng + " KAH220/MV1 #:1 (Pick #) 6. KAH221(E/S)/1645, 15 ng + " KAH221/MV1 #:1 (Pick #) 7. KAH222(E/S)/2614, 24 ng + " KAH222/MV1 #:1 (Pick #) 8. KAH223(E/S)/1537, 14 ng + " KAH223/MV1 #:1 (Pick #) 9. KAH224(E/S)/1837, 17 ng + " KAH224/MV1 #:1 (Pick #) 10. MV1(E/X)/ 25 ng     1 2 3 4 5 6 7 8 9 10 Insert DNA 3.0 1.4 1.8 1.6 0.5 0.6 1.1 1.1 0.7 --- Vector DNA 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 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 T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 dH2O 0.6 2.2 1.8 2.0 3.1 3.0 2.5 2.5 2.9 3.6   10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL --> Add 30 DH5α Turbo; plate on 100 μg/mL amp