Kozane - a primer design tool for site-directed mutagenesis

Kozane is a web tool that has been developed to make primer design for site-directed mutagenesis as seamless as possible. It should work for any method that relies on primers with the mismatch in the middle of the oligo, such as SAMURAI, Quikchange®, PFunkel and One-pot Saturation Mutagenesis. Simply fill in the form below to generate quality-checked primers with matching melting temperatures. In the next step, a single click can copy the selected primers for bulk input and bring you directly to the primer vendor of your choice. Check out the instructions in the upper right corner for more details.

1. Specify template and mutations

Template DNA sequence Enter the nucleotide sequence for the region you want to mutate.

Custom mutations

Alanine scan

Deep mutational scan

Custom scan

Enter nucleotide positions

Surface mutations

With the "Custom mutations" option, enter the amino acid positions and the mutations you want in the field below.

Enter mutations and their amino acid positions

Input help

Click the 'Input help' button to the right for further instructions and advance options.

Upstream DNA sequence Only required if mutations are located at beginning of the template sequence. Fifty bases are generally enough.

Downstream DNA sequence Only required if mutations are located towards the end of the template sequence. Fifty bases are generally enough.

2. Set primer properties

Min Tm: Set minimum allowed primer Tm

Max Tm: Set maximum allowed primer Tm

Max length primer: Set max length of primer.

Min flanking positions: Set minimum number of complementary bases on each side of the mismatch.

Make reverse primers

Make forward primers

Make both

Direction of selected primers

3. Choose ordering alternatives

Select primer provider: Select primer vendor

Primer name All primer names will start with whatever is in this field

5' ext / overhang Optional 5' extension or overhang on all primers