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Team:EPF-Lausanne/Protocols/Site-specific mutagenesis
From 2011.igem.org
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'''Purpose''': induce site-specific mutations in a gene, contained on a plasmid. | '''Purpose''': induce site-specific mutations in a gene, contained on a plasmid. | ||
| - | Site induced mutagenesis is a method to induce site-specific substitutions, deletions, or insertions in a plasmid. It is carried out using a PCR-like reaction, with two primers: one for the sense strand, one for the antisense strand. Both primers overlap the same region, and contain the desired mutations | + | Site induced mutagenesis is a method to induce site-specific substitutions, deletions, or insertions in a plasmid. It is carried out using a PCR-like reaction, with two primers: one for the sense strand, one for the antisense strand. Both primers overlap the same region, and contain the desired mutations. |
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The principle of site-directed mutagenesis is explained in the manual, linked above. | The principle of site-directed mutagenesis is explained in the manual, linked above. | ||
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The primers are designed using Agilent's online tool: [https://www.genomics.agilent.com/CollectionOverview.aspx?PageType=Application&SubPageType=ApplicationOverview&PageID=111|QuickChange Primer Design Program] (requires login; use the iGEM EPFL gmail address. Ask Doug for the password). | The primers are designed using Agilent's online tool: [https://www.genomics.agilent.com/CollectionOverview.aspx?PageType=Application&SubPageType=ApplicationOverview&PageID=111|QuickChange Primer Design Program] (requires login; use the iGEM EPFL gmail address. Ask Doug for the password). | ||
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| + | According to the [https://www.genomics.agilent.com/files/manual/210518.pdf|Agilent manual] for the kit we use, the primers are designed with the following constraints: | ||
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| + | * Length between 25 and 45 bases, ideally. | ||
| + | * Melting temperature above 78° C. | ||
| + | * Mutation near the middle of the primers, with 10 to 15 bases on each side. | ||
| + | * Ideally, have a GC content of at least 40%, and terminate with one or more G or C bases. | ||
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| + | The online tool ensures the constraints are met, all that is needed is to choose the site and type of mutation. | ||
The easiest way to design primers is to copy the wild-type sequence into the sequence, then "upload and translate" it. A little clarification to the provided documentation: when the sequence is uploaded, check boxes appear below a set of radio-buttons. They list the amino acids, translated from the provided sequence. Check up to seven positions, then choose what substitutions to operate using the drop-down menus ''above''. | The easiest way to design primers is to copy the wild-type sequence into the sequence, then "upload and translate" it. A little clarification to the provided documentation: when the sequence is uploaded, check boxes appear below a set of radio-buttons. They list the amino acids, translated from the provided sequence. Check up to seven positions, then choose what substitutions to operate using the drop-down menus ''above''. | ||
Revision as of 09:06, 15 July 2011
Site-specific mutagenesis of tetR
Back to protocols.
Purpose: induce site-specific mutations in a gene, contained on a plasmid.
Site induced mutagenesis is a method to induce site-specific substitutions, deletions, or insertions in a plasmid. It is carried out using a PCR-like reaction, with two primers: one for the sense strand, one for the antisense strand. Both primers overlap the same region, and contain the desired mutations.
The principle of site-directed mutagenesis is explained in the manual, linked above.
Primer Design
The primers are designed using Agilent's online tool: Primer Design Program (requires login; use the iGEM EPFL gmail address. Ask Doug for the password).
According to the manual for the kit we use, the primers are designed with the following constraints:
- Length between 25 and 45 bases, ideally.
- Melting temperature above 78° C.
- Mutation near the middle of the primers, with 10 to 15 bases on each side.
- Ideally, have a GC content of at least 40%, and terminate with one or more G or C bases.
The online tool ensures the constraints are met, all that is needed is to choose the site and type of mutation.
The easiest way to design primers is to copy the wild-type sequence into the sequence, then "upload and translate" it. A little clarification to the provided documentation: when the sequence is uploaded, check boxes appear below a set of radio-buttons. They list the amino acids, translated from the provided sequence. Check up to seven positions, then choose what substitutions to operate using the drop-down menus above.
Primers are then ordered from XXX who? XXX.
Mutation reaction
To be continued, once the primers are received...
