User:Karmella Haynes/Notebook/BioBrick cloning/2011/05/23

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05/23/11

  • ✓ Miniprep digests: KAH182(4)
  • ✓ Assemblies: KAH183, 184



Minipreps
> Check with E/P digests

Reagent Volume Expected:
1-4. KAH182 = 1669
Hover name
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

  1. KAH183: FC7/(E/S)/708 ✓ + (1) KAH182/(E/X)/4869
  2. KAH184: HPK/(E/S)/516 ✓ + "

> Digests (Fermentas FD)

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) 0.143 1.76 7.1


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. FC7(E/S)/708, 7 ng + KAH182(E/X)/4869, 25 ng KAH183 10:1 (Pick 2)
2. HPK(E/S)/516, 5 ng + " KAH184 2:1 (Pick 4)
3. KAH182(E/X)/ 25 ng  
  1 2 3
Insert DNA 1.0 1.0 ---
Vector DNA 3.5 3.5 3.5
2x lgn buf (Roche) 5.5 5.5 5.5
T4 ligase (NEB) 1.0 1.0 1.0
dH2O --- --- 1.0
  11 μL 11 μL 11 μL

---> Add 11 μL to 30 μL DH5α Turbo


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