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MutantMaker chooses oligonucleotides for Site Directed Mutagensis that will not
only mutate the codon to a target amino acid, but also ensure that the mutation
creates or destroyes a restriction site. By doing this, screening mutants becomes
much easier: instead of performing costly sequencing reactions, simply perform a digest.
If the restriction site is created or destroyed (easily detectable by comparison to
an experimental control), the screened candidate contains the desired mutation.
Follow the following steps to find an oligonucleotide for your mutation:
- Paste your DNA sequence (no spaces) into the field at the top of the MutantMaker window.
- Drag the codon selector back and forth to select the codon you want to mutate.
- Under "Settings", choose the amino acid you want to mutat the selected codon to.
- Push "Find Candidates".
- You're done.
MutantMaker can optionally change codons flanking the 5' or 3' side of the
selected mutation. Although the codons will change, the amino acid they encode
will not. Choose how many flanking codons you would like MutantMaker to attempt
to mutate by selecting values from the popup menus labeled "5' Codon Spread" and
"3' Codon Spread".
MutantMaker will only screen potential oligonucleotides with restriction
enzymes that are avaialble under the "Restriction Enzymes" tab. Only enzymes
with a checkbox next to them will be used. To bulk-select or bulk-unselect enzymes, simply
select them from the "Use" or "Don't Use" menus avaialble within "Options".
For example, to select all commercially avaialble 6 cutters, perform these steps:
- Choose Options → Don't use → All
- Choose Options → Use → 6 Cutters
- Choose Options → Don't Use → Not Commercially Available