User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/31

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Site Directed Mutagenesis of BD002 Ligation (Second Try)

  • Ligate The Sticky Ends to Make Circular DNA
Sample type Tube 1 Tube 2 (-) Ctrl
DNA Conc. (ng) 10ng 30ng 50ng 10ng 30ng 50ng ---
DNA μL 1μL 3μL 5μL 1μL 3μL 5μL 5μL
T4 Ligase 1.0 μl 1.0μL 1.0μL 1.0μL 1.0μL 1.0μL 0.0μL
2X Roche Buffer 5.00 μl 5.00 μL 6.00 μL 5.00 μL 5.00 μL 6.00 μL 5.00 μL
dH2O 3.00 μl 1.00 μl --- 3.00 μL 1.00 μL --- ---
Total 10.0 μl 10.0 μl 12.0 μl 10.0 μl 10.0 μl 12.00 μL 10.00 μL
Incubate at room temperature for 30 minutes.
Transformation result 6 Colonies 12 Colonies 100s of Colonies and Satellite colonies 19 Colonies 100s Colonies and Satellite Colonies 100s Colonies and Satellite Colonies No Colony
  • Fast transformation protocol in DH5α-T. Add 30 μL of cells to each sample and incubate on ice for 30 minutes.
  • Grow on AMP agar plates and incubate overnight at 37°C.
  • Take 3 colonies from 1 and 2 and 4 colonies from plate 4 for Colony PCR using mutagenesis primers for linearization of the plasmids.