User:Karmella Haynes/Notebook/BioBrick cloning/2011/05/24

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05/24/11

  • ✓ Cultures/minipreps/digests: 2 mL cultures for KAH183(2); KAH184(4)



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1,2. BB 1 = 2383
3-6. BB2 = 2191
Miniprep digest 5/24/11
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.


--> KAH184 not successful. Re-do with higher ratio of insert


Assemblies
--> Re-build KAH184

  1. KAH184: HPK/(E/S)/516 + KAH182/(E/X)/4869


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. HPK(E/S)/516, 15 ng + KAH182(E/X)/4869, 25 ng KAH183 10:1 (Pick 2)
2. KAH182(E/X)/ 25 ng  
  1 2 3
Insert DNA 3.0 ---
Vector DNA 3.5 3.5
2x lgn buf (Roche) 7.5 5.5
T4 ligase (NEB) 1.0 1.0
dH2O --- 3.0
  15 μL 15 μL 15 μL

---> Add 15 μL to 40 μL DH5α Turbo



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