User:Karmella Haynes/Notebook/BioBrick cloning/2011/01/19

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01/19/11 ✓ Assemblies: KAH179, 180; KAH176, 177, 178/ MV8 (AY2 zeo vector) Assemblies --> Clone Repressor expression constructs in the rev direction to test effectiveness of polyA BioBrick KAH179: (1) FC7/(E/S)/714 + (2) KAH175/(E/X)/~5421 KAH180: HPK/(E/S)/516 ✓ + " KAH176/MV8 fwd: (3) KAH176/(E/S)/3191 + (6) MV8/(E/X)/4594 KAH177/MV8 fwd: (4) KAH177/(E/S)/3384 + " KAH178/MV8 fwd: (5) KAH178/(E/S)/3395 + " KAH176/MV8 rev: KAH176/(E/S)/3191 + (7) MV8/(E/S)/4594 KAH177/MV8 rev: KAH177/(E/S)/3384 + " KAH178/MV8 rev: KAH178/(E/S)/3395 + " > Digests (Fermentas FD) --> Cut KAH176, 177, 178 w/ E/S/ScaI Reagent 1,2,6 3,4,5,7 DNA (plasmid) up to 25 μL up to 24 μL 10x buffer 3.0 3.0 enzyme 1 1.0 1.0 enzyme 2 1.0 1.0 enzyme 3 --- 1.0 dH2O --- ---   30 μL --> 37°C/ ~30 min. > Measure conc.'s Sample OD260 260/280 ng/μL 1. FC7 (E/S) 0.391 1.91 19.6 2. KAH175 (E/X) 0.262 2.10 13.1 3. KAH176 (E/S) 0.332 2.02 16.6 4. KAH177 (E/S) 0.279 2.25 13.9 5. KAH178 (E/S) 0.314 2.16 15.7 6. MV8 (E/X) 0.834 1.80 41.7 6. MV8 (E/S) 0.843 1.80 42.1 > Ligations Ligation Plate results (lig : neg crtl) 01/20/11 1. FC7(E/S)/714, 6.6 ng + KAH175(E/X)/~5421, 25 ng KAH179 #:1 (Pick 2) 2. HPK(E/S)/516, 4.8 ng + " KAH180 #:1 (Pick 2) 3. KAH175(E/X)/ 25 ng   4. KAH176(E/S)/3191, 42 ng + MV8(E/X)/4594, 30 ng KAH176/MV8 #:1 (Pick 2) 5. KAH177(E/S)/3384, 44 ng + " KAH177/MV8 #:1 (Pick 2) 6. KAH178(E/S)/3395, 44 ng + " KAH178/MV8 #:1 (Pick 2) 7. MV8(E/X)/ 30 ng   8. KAH176(E/S)/3191, 42 ng + MV8(E/S)/4594, 30 ng KAH176/MV8 #:1 (Pick 2) 9. KAH177(E/S)/3384, 44 ng + " KAH177/MV8 #:1 (Pick 2) 10. KAH178(E/S)/3395, 44 ng + " KAH178/MV8 #:1 (Pick 2) 11. MV8(E/S)/ 30 ng     1 2 3 4 5 6 7 8 9 10 11 Insert DNA 1.0 1.0 --- 3.3 3.3 3.3 --- 3.3 3.3 3.3 --- Vector DNA 2.0 2.0 2.0 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7 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 1.0 1.0 2.0 --- --- --- 3.3 --- --- --- 3.3   10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL