Team:UTP-Software/SoftwareTool

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(Created page with "{{Team:UTP-Software/utptemplate2}} == '''S<sup>2</sup>MT Tutorial''' == ''Write here the tutorial.'' == Next S<sup>2</sup>MT version == == Project Details == ''Tell us mo...")
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== Project Details ==
== Project Details ==
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''Tell us more about your project.  Give us background.  Use this is the abstract of your project. Be descriptive but concise (1-2 paragraphs).''
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==== Steps Followed to create the primers: ====
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<div align="justify"><ul>
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<li> Both the mutagenic primers must contain the desired mutation and anneal to the same sequence on opposite strands of the plasmid.</li>
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<li> Primers should be between 25 and 45 bases in length, and the melting temperature (Tm) of the primers should be greater than or equal to 78°C. The following formula is commonly used for estimating the Tm of primers:</li>
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  Tm = 81.5 + 0.41(%GC) − 675 / N - where N is the primer length in base pairs.
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<li> The desired mutation (deletion or insertion) should be in the middle of the primer with ~10–15 bases of correct sequence on both sides.</li>
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<li> The primers optimally should have a minimum GC content of 40% and should terminate in one or more C or G bases.</li>
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<li> Primers need not be 5´ phosphorylated but must be purified either by fast polynucleotide liquid chromatography (FPLC) or by polyacrylamide gel electrophoresis (PAGE). Failure to purify the primers results in a significant decrease in mutation efficiency.</li>
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<li> It is important to keep primer concentration in excess. Stratagene suggests varying the amount of template while keeping the concentration of the primer constantly in excess.</li></ul></div>

Revision as of 11:24, 26 September 2012

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S2MT Tutorial

Write here the tutorial.


Next S2MT version

Project Details

Steps Followed to create the primers:

  • Both the mutagenic primers must contain the desired mutation and anneal to the same sequence on opposite strands of the plasmid.
  • Primers should be between 25 and 45 bases in length, and the melting temperature (Tm) of the primers should be greater than or equal to 78°C. The following formula is commonly used for estimating the Tm of primers:
    • Tm = 81.5 + 0.41(%GC) − 675 / N  - where N is the primer length in base pairs.
  • The desired mutation (deletion or insertion) should be in the middle of the primer with ~10–15 bases of correct sequence on both sides.
  • The primers optimally should have a minimum GC content of 40% and should terminate in one or more C or G bases.
  • Primers need not be 5´ phosphorylated but must be purified either by fast polynucleotide liquid chromatography (FPLC) or by polyacrylamide gel electrophoresis (PAGE). Failure to purify the primers results in a significant decrease in mutation efficiency.
  • It is important to keep primer concentration in excess. Stratagene suggests varying the amount of template while keeping the concentration of the primer constantly in excess.
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