Team:UTP-Software/SoftwareTool

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== Project Details ==
== Project Details ==
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==== S<sup>2</sup>MT in detail ====
==== Protocol Followed to design the primers:<sup>[1]</sup> ====
==== Protocol Followed to design the primers:<sup>[1]</sup> ====
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Revision as of 11:34, 26 September 2012

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S2MT Tutorial

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Project Details

S2MT in detail

Protocol Followed to design the primers:[1]

  • 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.

References

[1]. Stratagene. QuikChange™ Site-Directed Mutagenesis Kit. Catalog #200518. Revision #108005h. Available in: [http://web.physics.ucsb.edu/~deborah/pro/pro_pdf/Stratagene%20QuikChange.pdf].

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