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

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06/06/11

  • ✓ Assemblies: full miR sensors w/ different promoters/ binding site copy numbers
  • ✓ DH5α Turbo chemically competent cell prep: streak LB (plain) plates with left-over cells w/o DNA (from transformations)



Assemblies

  1. KAH186/196: KAH186/(E/S)/2025 ✓ + (1) KAH196/(E/X)/7806
  2. KAH184/196: KAH184/(E/S)/2191 ✓ + "
  3. KAH187/196: KAH187/(E/S)/2269 ✓ + "
  4. KAH188/196: KAH188/(E/S)/2124 ✓ + "
  5. KAH189/196: KAH189/(E/S)/2296 ✓ + "
  6. KAH190/196: KAH190/(E/S)/2368 ✓ + "
  7. KAH191/196: KAH191/(E/S)/2223 ✓ + "
  8. KAH183/196: KAH183/(E/S)/2383 ✓ + "
  9. KAH192/196: KAH192/(E/S)/2461 ✓ + "
  10. KAH186/177: KAH186/(E/S)/2025 ✓ + KAH177/(E/X)/7978 ✓
  11. KAH187/177: KAH187/(E/S)/2269 ✓ + "
  12. KAH188/177: KAH188/(E/S)/2124 ✓ + "
  13. KAH189/177: KAH189/(E/S)/2296 ✓ + "
  14. KAH190/177: KAH190/(E/S)/2368 ✓ + "
  15. KAH191/177: KAH191/(E/S)/2223 ✓ + "
  16. KAH192/177: KAH192/(E/S)/2461 ✓ + "
  17. KAH186/198: KAH186/(E/S)/2025 ✓ + (2) KAH198/(E/X)/8050
  18. KAH184/198: KAH184/(E/S)/2191 ✓ + "
  19. KAH187/198: KAH187/(E/S)/2269 ✓ + "
  20. KAH188/198: KAH188/(E/S)/2124 ✓ + "
  21. KAH189/198: KAH189/(E/S)/2296 ✓ + "
  22. KAH190/198: KAH190/(E/S)/2368 ✓ + "
  23. KAH191/198: KAH191/(E/S)/2223 ✓ + "
  24. KAH183/198: KAH183/(E/S)/2383 ✓ + "
  25. KAH192/198: KAH192/(E/S)/2461 ✓ + "


> Digests (Fermentas FD)

Reagent Volume   Cloning digest 6/06/11
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. KAH196 (E/X) 1.617 1.88 80.9
2. KAH198 (E/X) 1.353 1.85 67.6


> Ligations

Ligation Plate results (lig : neg crtl) 06/07/11
1-9. KAH###(E/S)/~2200, 11 ng + KAH196(E/X)/7806, 20 ng Notes: >10:1 for all plates
10. KAH196(E/X)/ 25 ng  
11-17. KAH###(E/S)/~2200, 11 ng + KAH177(E/X)/7978, 20 ng Notes: >10:1 for all plates
18. KAH177(E/X)/ 25 ng  
19-27. KAH###(E/S)/~2200, 11 ng + KAH198(E/X)/8050, 20 ng Notes: >10:1 for all plates
28. KAH198(E/X)/ 25 ng  
  1-9 10 11-17 18 19-27 28
Insert DNA 3.0 --- 3.0 --- 3.0 ---
Vector DNA 0.25 0.25 1.0 1.0 0.3 0.3
2x lgn buf (Roche) 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
dH2O 0.75 3.75 --- 3.0 0.7 3.7
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL

--> Make master mix w/ lign. buffer, vector DNA; add insert DNA to individual tubes; add ligase to master mix and quickly aliquot
--> Add rxn. to 25 μL DH5α Turbo cells



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