User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/27

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12/27/09

  • Re-transform: KAH93/V0120; JDS42, 43
  • Assemblies: KAH136; KAH137, 138 (do-over)

Assemblies

  1. KAH136: (1) PEST/(S/P)/3734 + EED/(X/P)/1401 ✓
  2. KAH137: KAH78/(E/S)/3652 ✓ + KAH134/(E/X)/4600 ✓
  3. KAH138: KAH79/(E/S)/3652 ✓ + "

> Digests (Fermentas FD)

Reagent Volume   Cloning digests 12/16/09
30 μL/lane, 1% agarose
DNA (plasmid) 5.0
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O 20.0
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. PEST(S/P) 0.197 2.18 9.9


> Ligations

Ligation Plate results (lig : neg ctrl) 12/28/09
1. EED(X/P)/1401, 15 ng + PEST(S/P)/3734, 20 ng KAH136 10:1 (Pick 2)
2. PEST(S/P)/ 20 ng + "  
3. KAH78(E/S)/3652, 24 ng + KAH134(E/X)/4600, 10 ng KAH137 2:1 (Pick 4)
4. KAH79(E/S)/3724, 24 ng + " KAH138 2:1 (Pick 4)
5. KAH134(E/X)/ 10 ng  

--> For KAH137 & 138, use less vector and 3:1 insert to minimize background

  1 2 3 4 5
Insert DNA 0.5 --- 1.2 1.2 ---
Vector DNA 2.0 2.0 0.8 0.8 0.8
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0
dH2O 1.5 2.0 2.0 2.0 3.2
  10μL 10μL 10μL 10μL 10μL --> R.T./ ~15 min.; Add 25 μL Z-DH5α


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