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

  1. KAH179: (1) FC7/(E/S)/714 + (2) KAH175/(E/X)/~5421
  2. KAH180: HPK/(E/S)/516 ✓ + "
  3. KAH176/MV8 fwd: (3) KAH176/(E/S)/3191 + (6) MV8/(E/X)/4594
  4. KAH177/MV8 fwd: (4) KAH177/(E/S)/3384 + "
  5. KAH178/MV8 fwd: (5) KAH178/(E/S)/3395 + "
  6. KAH176/MV8 rev: KAH176/(E/S)/3191 + (7) MV8/(E/S)/4594
  7. KAH177/MV8 rev: KAH177/(E/S)/3384 + "
  8. 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