"
Team:EPF-Lausanne/Protocols/Site-specific mutagenesis
From 2011.igem.org
(Created page with "{{:Team:EPF-Lausanne/Templates/ProtocolHeader|title=Site-specific mutagenesis of tetR}} {{:Team:EPF-Lausanne/Templates/Footer}}") |
|||
| Line 1: | Line 1: | ||
{{:Team:EPF-Lausanne/Templates/ProtocolHeader|title=Site-specific mutagenesis of tetR}} | {{:Team:EPF-Lausanne/Templates/ProtocolHeader|title=Site-specific mutagenesis of tetR}} | ||
| + | |||
| + | |||
| + | '''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. 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: | ||
| + | |||
| + | * 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. | ||
| + | |||
| + | == Primer Design == | ||
| + | |||
| + | 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 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... | ||
{{:Team:EPF-Lausanne/Templates/Footer}} | {{:Team:EPF-Lausanne/Templates/Footer}} | ||
Revision as of 09:02, 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. 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.
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).
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...
