Difference between revisions of "User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/11/01"

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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.  
 
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 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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Here we plan to mutate the BSMBI cut site: CGTCTC to CGTTTC
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Design Primers:
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<span style="color:#A9A9A9">gca</span> <span style="color:#FF0000">GCTCTTC</span> '''G''' <span style="color:#228B22">'''T'''</span>TC
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Forward Primer: 5’ gca GCTCTTC G TTC  25 pbs of top right
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Reverse Primer 5’ gca GCTCTTC G GAAACG 25 pbs of bottom left
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*'''[[User:Behzad Damadzadeh|Behzad Damadzadeh]] 13:34, 4 November 2013 (EST)''':
 
*'''[[User:Behzad Damadzadeh|Behzad Damadzadeh]] 13:34, 4 November 2013 (EST)''':

Revision as of 12:33, 4 November 2013

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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:

gca GCTCTTC G TTC

Forward Primer: 5’ gca GCTCTTC G TTC 25 pbs of top right

Reverse Primer 5’ gca GCTCTTC G GAAACG 25 pbs of bottom left