User:Karmella Haynes/Notebook/BioBrick cloning/2011/05/20

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05/20/11

  • ✓ Assembly: Remove PEST from CFP reporter gene; KAH181
  • ✓ Retransform: KAH57



Assemblies

  1. KAH181: Kozak/(S/P)/~3200 ✓ + (1) KAH57/(X/P)/1426

> Digests (Fermentas FD)

Reagent Volume   File:KAH 052011 gel1.tif
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. KAH57 (X/P) 0.543 1.81 27.2


> Ligations

Ligation Plate results (lig : neg crtl) 05/21/11
1. KAH57(X/P)/1426, 22 ng + Kozak(S/P)/~3200, 25 ng KAH181 >10:1 (Pick 2)
2. Kozak(S/P)/ 25 ng  
  1 2
Insert DNA 1.0 ---
Vector DNA 0.5 0.5
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 2.5 3.5
  10 μL 10 μL

--> Add 10 μL lgn rxn. to 30 μL Dh5α Turbo
--> Also do retransformation of KAH57