User:Karmella Haynes/Notebook/BioBrick cloning/2011/07/13

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07/13/11

  • ✓ Cultures/minipreps: KAH213-218, KAH207-209/V0200 (2 each)
  • ✓ Assemblies: miR-luc sensor set with 177/MV8 vector (re-build, see 6/7/11)



Minipreps
> Check KAH213-218 with E/P
> Check KAH207-209/V0200 with N/S

Reagent Volume Expected:
V0120 = ~3200
1,2. KAH213 = 2834
3,4. KAH214 = 1757
5,6. KAH215 = 2057
7,8. KAH216 = 2590
9,10. KAH217 = 1513
11,12. KAH218 = 1813


V0200 = 4442
13,14. KAH207/V0200 = 1836
15,16. KAH208/V0200 = 1884
17,18. KAH209/V0200 = 1932
Miniprep digests
15 μL/lane; 1% agarose

Miniprep digests
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.

Assemblies
--> Last time (6/06/11), may have used too much insert. Use calculated amount this time
--> Use 177/MV8(E/X) 1/25/11. Was successful with 183-177/MV8 assembly (see 7/09/11)

  1. KAH186-177/MV8: KAH186/(E/S)/2025 ✓ + KAH177/MV8/(E/X)/7978 ✓
  2. KAH184-177/MV8: KAH187/(E/S)/2269 ✓ + "
  3. KAH187-177/MV8: KAH187/(E/S)/2269 ✓ + "
  4. KAH188-177/MV8: KAH188/(E/S)/2124 ✓ + "
  5. KAH189-177/MV8: KAH189/(E/S)/2296 ✓ + "
  6. KAH190-177/MV8: KAH190/(E/S)/2368 ✓ + "
  7. KAH191-177/MV8: KAH191/(E/S)/2223 ✓ + "
  8. KAH192-177/MV8: KAH192/(E/S)/2461 ✓ + "


> Ligations

Ligation Plate results (lig : neg crtl) 07/14/11
1-8. KAH###(E/S)/~2200, 11 ng + KAH177/MV8(E/X)/7978, 20 ng Notes: >10:1 for all plates
9. KAH177(E/X)/ 20 ng  
  1 2 3 4 5 6 7 8 9
Insert DNA 1.0 1.0 1.5 1.0 1.0 0.5 1.0 1.0 ---
Vector DNA 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
2x lgn buf (Roche) 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
dH2O 2.0 2.0 1.5 2.0 2.0 2.5 2.0 2.0 3.0
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL

--> Add to 30 μL DH5α Turbo; plate on 100 μg/mL amp


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