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 20 μL of nuclease free water, then cut with E/P and purified from the gel.
  • Ligations
Ligation </font>
1. E. coli DH5α-T KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng KAH111/C-His 3:1 No Colonies
2. E. coli BL-21 KAH111(E/P)/size, 16 ng + C-His(E/P)/3600, 26ng KAH111/C-His 3:1 No Colonies
1. E. coli DH5α-T KAH112(E/P)/size, 22.8 ng + C-His(E/P)/3600, 26ng KAH112/C-His 3:1 No Colonies
2. E. coli BL-21 KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng KAH112/C-His 3:1 No Colonies
3. C-His(E/P)/ 25 ng
  1 2 3
Insert DNA 3.12 3.12 ---
Vector DNA 2.94 2.94 2.94
2x lgn buf (Roche) 7.06 7.06 5.0
T4 ligase (NEB) 1.0 1.0 1.0
dH2O 0 0 0.88
  14.12 μL 14.12 μL 10μl