User:Karmella Haynes/Notebook/BioBrick cloning/2010/03/15

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03/15/10

  • ✓ Assembly: KAH142 - DPRE+YFP reporter (KAH96)



Assemblies

  1. KAH142: (1) DPRE/(E/S)/1740 + (2) KAH96/(E/X)/~6025

> Digests (Fermentas FD)

Reagent Volume   File:KAH 031510 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. DPRE (E/S) 0.863 1.93 43.2
2. KAH96 (E/X) 2.379 1.85 118.9


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. DPRE(E/S)/1740, 14 ng + KAH96(E/X)/6025, 25 ng KAH142 3:1 (Pick 6)
2. KAH96(E/X)/ 25 ng >100 colonies
  1 2
Insert DNA 0.8 ---
Vector DNA 0.2 0.2
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 3.0 3.8
  10 μL 10 μL

--> R.T./ 10 min., Add to 50 μL Z-DH5α