User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/01/12

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01/12/2013

Ligation of KAH111, KAH112 and C-His vector in DH5α-T and BL21

  • The 4 μL of C-His Vector from stock is diluted with 16 μL of nuclease free water, then cut with E/P and purified from the gel.
  • Ligations
Ligation
1. E. coli BL-21 KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng KAH111/C-His 3:1 No Colonies
2. E. coli DH5α-T KAH111(E/P)/size, 16 ng + C-His(E/P)/3600, 26ng KAH111/C-His 3:1 No Colonies
3. E. coli BL-21 KAH112(E/P)/size, 22.8 ng + C-His(E/P)/3600, 26ng KAH112/C-His 3:1 No Colonies
4. E. coli DH5α-T KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng KAH112/C-His 3:1 No Colonies
5. C-His(E/P)/ 26 ng (Control Plate)
  • Calculations are for the ng of insert we need to get a 3:1 ratio of insert molecules to 50 ng vector molecules
  1 2 3 4 5
Insert DNA 2.34 2.34 2.05 2.05 ---
Vector DNA 1.92 1.92 1.92 1.92 1.92
2x lgn buf (Roche) 5.26 5.26 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0
dH2O 0 0 0.03 0.03 2.08
  10.52 μL 10.52 μL 10μl 10μL 10μL
  • The incubation time for the ligation process was 30min at room temperature.
  • Fast transformation, 30 min on ice.
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