User:Wesley J. Houston/Notebook/MAP/2013/04/01

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4/1/13

  • Completed 2 ligations of KAH201 (S/P)/3403 + KAH182 (X/P)/1675
    • First ligation used KAH201 (S/P)/3403 and KAH182 (X/P) 1675 made on 3/26/13
    • Second ligation used the same KAH182 (X/P) 1675 and KAH201 (S/P)/3403 made on 2/8/13

> Ligation 1

  1 2
Insert DNA (KAH182) 3.5 ---
Vector DNA (KAH201) 1.0 1.0
2x lgn buf (Roche) 5.5 5.5
T4 ligase (NEB) 1.0 1.0
dH2O 0.0 3.5
  11 μL 11 μL
  • Follow quick transformation protocol
  • Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar
Ligation Plate results (lig : neg crtl)04/01/13: Ligation 1
1. KAH201 (S/P)/3403, 25 ng + KAH182 (X/P)/1675, 26 ng 0
2. KAH201 (S/P)/3403, 25 ng 0


> Ligation 2

  1 2
Insert DNA (KAH182) 3.5 ---
Vector DNA (KAH201) 0.7 0.7
2x lgn buf (Roche) 5.2 5.2
T4 ligase (NEB) 1.0 1.0
dH2O 0.6 4.1
  11 μL 11 μL
  • Follow quick transformation protocol
  • Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar
Ligation Plate results (lig : neg crtl)04/01/13: Ligation 2
1. KAH201 (S/P)/3403, 25 ng + KAH182 (X/P)/1675, 26 ng ~40
2. KAH201 (S/P)/3403, 25 ng 0
  • Ligation 2 appears successful, with ~40 colonies in the ligation plate and 0 colonies in the negative control plate. Results of the ligation
    • The next step will be to confirm the assembly, to be completed on 4/4/13


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