Team:UTP-Software/SoftwareTool

From 2012.igem.org

Revision as of 11:32, 26 September 2012 by Egaruz89 (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

Home	Team & Attributions	Project	S²MT	Tutorial	Biosinergia	Notebook	Human Practices	Safety	Sponsors

S²MT Tutorial

Writethe tutorial here.

Next S²MT Version

Project Details

Steps 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].