"
Team:EPF-Lausanne/Protocols/Site-specific mutagenesis
From 2011.igem.org
| Line 4: | Line 4: | ||
'''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 | + | ''Site-specific mutagenesis'' or ''site-directed 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 more detail in the [https://www.genomics.agilent.com/files/manual/210518.pdf|Agilent manual] for their mutagenesis kits. | ||
| + | |||
| + | Once all components (primers, buffers, culture media...) are available, the procedure should take approximately one half-day, followed by an overnight culture, then a miniprep, that should take an hour or so. The procedure is outlined as follows: | ||
| + | |||
| + | * '''Thermal Cycling''': extension reaction to copy the template and induce mutations (PCR-like reaction) (1 hour). | ||
| + | * '''Digestion of template''': enzymes digest the template (unmutated by definition) to leave only the mutants (5 minutes). | ||
| + | * '''Transformation''' into competent cells: to repair nicks left in the plasmid by the extension reaction (1.5 hours). | ||
| + | * '''Overnight culture''': to amplify the DNA (1 night). | ||
| + | * '''Miniprep''': to recover the mutated and amplified DNA (1 hour). | ||
| - | |||
== Primer Design == | == Primer Design == | ||
Revision as of 13:43, 19 July 2011
Site-specific mutagenesis of tetR
Back to protocols.
Purpose: induce site-specific mutations in a gene, contained on a plasmid.
Site-specific mutagenesis or site-directed 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 more detail in the manual for their mutagenesis kits.
Once all components (primers, buffers, culture media...) are available, the procedure should take approximately one half-day, followed by an overnight culture, then a miniprep, that should take an hour or so. The procedure is outlined as follows:
- Thermal Cycling: extension reaction to copy the template and induce mutations (PCR-like reaction) (1 hour).
- Digestion of template: enzymes digest the template (unmutated by definition) to leave only the mutants (5 minutes).
- Transformation into competent cells: to repair nicks left in the plasmid by the extension reaction (1.5 hours).
- Overnight culture: to amplify the DNA (1 night).
- Miniprep: to recover the mutated and amplified DNA (1 hour).
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...
