User:Karmella Haynes/Notebook/BioBrick cloning/2011/05/31

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05/31/11

  • ✓ Assemblies: Re-build mIR sensor test constructs without insulators and with CFP-Reporter, no PEST



Assemblies
--> Old plasmids are very problematic. As long as I'm rebuilding, may as well update the initial miR sensor test experiments to match the current design
--> Build complete "matrix" of conditions:

  • Px (RFP-repressor promoter) = Ubc, HPK, CMV
  • Py (CFP-reporter promoter) = Ubc, HPK, CMV
  • Gal4 binding sites = 0, 5x, 10x

--> Differences from previous constructs: No HS4 insulators, final constructs in MV8, no PEST on CFP, polyA2 (Mara's M127)

  1. KAH185: KAH83/(E/S)/443 ✓ + 5xGal4/(E/X)/~3293 ✓
  2. KAH186: Ubc/(E/S)/344 ✓ + (1) KAH182/(E/X)/~4875
  3. KAH187: CMV/(E/S)/588 ✓ + "
  4. KAH188: KAH83/(E/S)/443 + "
  5. KAH189: KAH84/(E/S)/615 + "
  6. KAH190: KAH85/(E/S)/687 + "
  7. KAH192: (2) FC6/(E/S)/780 + "
  8. KAH193: Ubc/(E/S)/344 ✓ + KAH73/(E/X)/~5638 ✓
  9. KAH194: CMV/(E/S)/588 ✓ + "


> Digests (Fermentas FD)
--> Specific notes

Reagent Volume  
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. KAH182 (E/X) 0.144 1.71 7.2
2. FC6 (E/S) 0.505 1.58 25.2


> Ligations

Ligation Plate results (lig : neg crtl) 06/01/11
1. KAH83(E/S)/443, 7 ng + 5xGal4(E/X)/3293, 25 ng KAH185 3:1 (Pick 2)
2. 5xGal4(E/X)/ 25 ng  
3. Ubc(E/S)/344, 5 ng + KAH182(E/X)/3293, 25 ng KAH186 1:1 (Pick 2)
4. CMV(E/S)/588, 9 ng + " KAH187 1:1 (Pick 2)
5. KAH83(E/S)/443, 7 ng + " KAH188 1:1 (Pick 2)
6. KAH84(E/S)/615, 9 ng + " KAH189 1:1 (Pick 2)
7. KAH85(E/S)/687, 10 ng + " KAH190 1:1 (Pick 2)
8. FC6(E/S)/780, 12 ng + " KAH192 1:1 (Pick 2)
9. KAH182(E/X)/ 25 ng lots of colonies
10. Ubc(E/S)/344, 3 ng + KAH73(E/X)/5638, 25 ng KAH193 10:1 (Pick 2)
11. CMV(E/S)/588, 5 ng + " KAH194 10:1 (Pick 2)
12. KAH73(E/X)/ 25 ng  
  1 2 3 4 5 6 7 8 9 10 11 12
Insert DNA 2.0 --- 2.0 2.0 2.0 2.0 2.0 2.0 --- 2.0 2.0 ---
Vector DNA 0.5 0.5 2.0 2.0 2.0 2.0 2.0 2.0 2.0 0.5 0.5 0.5
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 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 1.0
dH2O 1.5 3.5 --- --- --- --- --- --- 2.0 1.5 1.5 3.5
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL