User:Karmella Haynes/Notebook/BioBrick cloning/2009/12/17

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12/17/09

  • ✓ Assemblies: re-do KAH117-119, 135, 136

Assemblies

  1. KAH117: KAH99/(E/S)/2913 ✓ + (1) KAH93/(E/X)/8039
  2. KAH118: KAH100/(E/S)/3085 ✓ + "
  3. KAH119: KAH101/(E/S)/3157 ✓ + "
  4. KAH135: KAH78/(E/S)/3652 ✓ + (2) KAH134/(E/X)/4600
  5. KAH136: KAH78/(E/S)/3652 ✓ + "

> Digests (Fermentas FD)
--> Re-do, do not phosphatase treat; use less DNA to ensure complete digestion

Reagent Volume   File:KAH 121709 gel1.tif
30 μL/lane, 1% agarose
DNA (plasmid) 5.0
10x buffer 3.0
enzyme 1 2.0
enzyme 2 2.0
dH2O 18.0
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. KAH93(E/X) 0.140 2.54 7.0
2. KAH134(E/X) 0.242 2.57 12.1


> Ligations

Ligation Plate results (lig : neg ctrl) 12/18/09
1. KAH99(E/S)/2913, 22 ng + KAH93(E/X)/8039, 30 ng KAH117 3 colonies (Pick 3)
2. KAH100(E/S)/3085, 23 ng + " KAH118 10:1 (Pick 4)
3. KAH101(E/S)/3157, 23 ng + " KAH119 1 colony (Pick 1)
4. KAH93(E/X)/ 30 ng 1 colony
5. KAH78(E/S)/3652, 41 ng + KAH134(E/X)/4600, 20 ng KAH135 1:1 (re-do)
6. KAH79(E/S)/3724, 41 ng + " KAH136 1:1 (re-do)
7. KAH134(E/X)/ 20 ng lots of colonies
  1 2 3 4 5 6 7
Insert DNA 0.7 0.7 0.7 --- 2.3 2.3 ---
Vector DNA 4.3 4.3 4.3 4.3 1.7 1.7 1.7
2x lgn buf (Roche) 6.0 6.0 6.0 6.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O --- --- --- 0.7 --- --- 2.3
  12μL 12μL 12μL 12μL 10μL 10μL 10μL --> R.T./ ~15 min.; Add 25 μL Z-DH5α