User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/11/01: Difference between revisions
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= | <div class="center" style="width: auto; margin-left: auto; margin-right: auto;">'''PCR Based Mutagenesis Protocol'''</div> | ||
''' | 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: | |||
*'''[[User:Behzad Damadzadeh|Behzad Damadzadeh]] 13:34, 4 November 2013 (EST)''': | |||
Revision as of 11:34, 4 November 2013
 PcTF Subcloning in E. coli
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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:
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