Overview of the Q5^® Site-Directed Mutagenesis Kit

The Q5 Site-Directed Mutagenesis Kit allows rapid site-specific mutagenesis of double-stranded plasmid DNA in less than 2 hours.

The kit utilizes the robust Q5 Hot Start High-Fidelity DNA Polymerase and custom mutagenic primers to create substitutions, deletions and insertions for a wide variety of plasmids up to at least 14 kb in length.

Primer design:

For best results, back-to-back primers should be designed using NEBaseChanger, our online primer design tool. Standard, non-phosphorylated primers can be used.  NEBaseChanger also calculates the annealing temperature for the specific mutagenic primers while accounting for the high-fidelity polymerase, Q5. If NEBaseChanger is not used to design primers, we recommend using a Tm+3 rule to calculate the annealing temperature.

Unlike kits that rely on linear amplification, primers designed for the Q5 SDM Kit should not overlap. Substitutions are created by incorporating the desired nucleotide change, or changes, in the center of the forward primer. At least 10 complementary nucleotides must be included on the 3-prime side of the mutation. The reverse primer is designed so that the 5-prime ends of the two primers anneal back-to-back.

Primer design: Deletions

Deletions are engineered by designing standard, non-mutagenic forward and reverse primers that flank the region to be deleted.

Primer design: Insertions

Insertions less equal to 6 nucleotides are incorporated into the 5-prime end of the forward primer, and the reverse primer should anneal back-to-back with the forward primer. Larger insertions are created by incorporating half of the desired insertion into the 5-prime ends of both primers. The maximum size of the insertion is largely dictated by oligonucleotide synthesis limitations.

Step One: Exponential amplification

Q5 Master Mix, primers and template are combined in a single tube, and exponential amplification occurs.

Step Two: Treatment and enrichment

Next, the PCR product is combined with a unique KLD Mix, comprised of a kinase, a ligase and DpnI, and the mix is incubated for 5 minutes. During this time, rapid phosphorylation and ligation occur, thereby circularizing the PCR product and removing the template.

Step Three: Transformation

The reaction is then transformed into chemically competent cells, such as NEB 5-alpha Competent E. coli. Our easy-to-use method results in a higher percentage of desired mutations, saving you precious screening time.

To learn more, visit neb.com/E0554.