User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/11/01

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PCR Based Mutagenesis Protocol

It is fast and efficient method using type IIs restriction enzymes. With designing a pair of mutagenesis primers that the mutated sequence is near and downstream of the recognition site of the type IIs restriction enzyme. PCR will linearize the plasmid with two blunt ends. Enzyme digestion will produce two sticky and complementary ends that will ligate together and generate the mutated plasmid. Here this method would be applied to mutate the BSMBI cut site within the hygromycine resistant gene of V0200 mammalian vector. Primers are designed for SapI restriction enzyme:

Here we plan to mutate the BSMBI cut site: CGTCTC to CGTTTC Design Primers:

  • Forward Primer: 5' gca GCTCTTC G TTC + 15 bps of top right strand
  • Reverse Primer: 5' gca GCTCTTC G GAAACG + 15 bps of bottom left strand

PCR Reactions

Reagents Volume Reaction Conditions
DNA Template (ng) 200
  • 95°C, 3 min.
  • [95°C, 30 sec.; 45°C, 40 sec.; 72°C, 6 min.] x35
  • 72°C, 3 min.
  • 4°C, ∞
Forward Promer (µL) 1
Reverse Primer (µL) 1
2X GoTag (µL) 50
dH2O (µL) 22.8
Tolal (µL) 50



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