User:Karmella Haynes/Notebook/BioBrick cloning/2011/02/27

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02/27/11

  • ✓ Assemblies: Pc-TF constructs into constitutive expression vector MV1 (pcDNA 3.1+neo/ CMV)



Assemblies

  1. KAH160/MV1: KAH160/(E/S)/1147 ✓ + (1) MV1/(E/X)/~5420
  2. KAH161/MV1: KAH161/(E/S)/1147 ✓ + "
  3. KAH162/MV1: KAH162/(E/S)/1147 ✓ + "
  4. KAH165/MV1: KAH165/(E/S)/991 ✓ + "
  5. KAH166/MV1: KAH166/(E/S)/991 ✓ + "
  6. KAH167/MV1: KAH167/(E/S)/991 ✓ + "
  7. KAH170/MV1: KAH170/(E/S)/955 ✓ + "


> Digests (Fermentas FD)

Reagent Volume  
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. MV1 (E/X) 0.863 2.12 43.1


> Ligations

Ligation Plate results (lig : neg crtl) 02/28/11
1. KAH160(E/S)/1147, 10.6 ng + MV1(E/X)/5420, 25 ng KAH160/MV1 10:1 (Pick 2)
2. KAH161(E/S)/1147, 10.6 ng + " KAH161/MV1 10:1 (Pick 2)
3. KAH162(E/S)/1147, 10.6 ng + " KAH162/MV1 10:1 (Pick 2)
4. KAH165(E/S)/991, 9.1 ng + " KAH165/MV1 10:1 (Pick 2)
5. KAH166(E/S)/991, 9.1 ng + " KAH166/MV1 10:1 (Pick 2)
6. KAH167(E/S)/991, 9.1 ng + " KAH167/MV1 10:1 (Pick 2)
7. KAH170(E/S)/955, 8.8 ng + " KAH170/MV1 10:1 (Pick 2)
8. MV1(E/X)/5420, 25 ng
  1 2 3 4 5 6 7 8
Insert DNA 3.4 3.4 3.4 3.4 3.4 3.4 3.4 ---
Vector DNA 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O --- --- --- --- --- --- --- 3.4
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL