User:Karmella Haynes/Notebook/BioBrick cloning/2011/06/03

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06/03/11

  • ✓ Assemblies: CFP-Reporter and RFP-Repressor genes into MV8 (11)



Assemblies

  1. KAH186/MV8: KAH186/(E/S)/2025 + MV8/(E/X)/4594 ✓
  2. KAH184/MV8: KAH184/(E/S)/2191 + "
  3. KAH187/MV8: KAH187/(E/S)/2269 + "
  4. KAH188/MV8: KAH188/(E/S)/2269 + "
  5. KAH189/MV8: KAH189/(E/S)/2124 + "
  6. KAH190/MV8: KAH190/(E/S)/2368 + "
  7. KAH191/MV8: KAH191/(E/S)/2223 + "
  8. KAH183/MV8: KAH183/(E/S)/2383 + "
  9. KAH192/MV8: KAH192/(E/S)/2461 + "
  10. KAH196/MV8: KAH196/(E/S)/3212 + "
  11. KAH198/MV8: KAH198/(E/S)/3456 + "


> Digests (Fermentas FD)
--> Digest KAH196, 198 w/ E/S/ScaI

Reagent Volume   Cloning digests 6/03/11
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. KAH186 (E/S) 0.286 2.14 14.3
2. KAH187 (E/S) 0.176 2.58 8.8
3. KAH188 (E/S) 0.376 2.34 18.8
4. KAH189 (E/S) 0.209 2.36 10.4
5. KAH190 (E/S) 0.520 1.93 26.0
6. KAH191 (E/S) 0.326 2.16 16.3
7. KAH192 (E/S) 0.265 2.81 13.2
8. KAH196 (E/S) 0.669 2.01 33.5
9. KAH198 (E/S) 0.687 1.98 34.4


> Ligations

Ligation Plate results (lig : neg crtl) 6/03/11
1. KAH186(E/S)/2025, 18 ng + MV8(E/X)/4594, 25 ng KAH186/MV8 >10:1 (Pick 2)
2. KAH184(E/S)/2191, 19 ng + " KAH184/MV8 >10:1 (Pick 2)
3. KAH187(E/S)/2269, 20 ng + " KAH184/MV8 >10:1 (Pick 2)
4. KAH188(E/S)/2124, 18 ng + " KAH184/MV8 >10:1 (Pick 2)
5. KAH189(E/S)/2296, 20 ng + " KAH184/MV8 >10:1 (Pick 2)
6. KAH190(E/S)/2368, 21 ng + " KAH184/MV8 >10:1 (Pick 2)
7. KAH191(E/S)/2223, 19 ng + " KAH184/MV8 >10:1 (Pick 2)
8. KAH183(E/S)/2383, 21 ng + " KAH184/MV8 >10:1 (Pick 2)
9. KAH192(E/S)/2461, 21 ng + " KAH184/MV8 >10:1 (Pick 2)
10. KAH196(E/S)/3212, 28 ng + " KAH184/MV8 >10:1 (Pick 2)
11. KAH198(E/S)/3456, 30 ng + " KAH184/MV8 >10:1 (Pick 2)
12. MV8(E/X)/ 25 ng  
  1-11 12
Insert DNA 3.5 ---
Vector DNA 0.5 0.5
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O --- 3.5
  10 μL 10 μL



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