KLD Mix for Back-to-Back Site-Directed Mutagenesis

KLD Mix Site-Directed Mutagenesis

The KLD Mix for Back to Back Site-Directed Mutagenesis from Civic Bioscience combines a unique blend of enzymes intended to be used for the site-directed mutagenesis strategy utilizing the back-to-back primer design. The enzymatic activities of T4 Polynucleotide Kinase, T4 DNA Ligase and DMT enzyme (DpnI) are combined and achieve optimal performance in the enhanced 5x reactional buffer. In contrast to competitor products, our KLD Mix is compatible with any High-Fidelity DNA Polymerase that generates strictly blunt end PCR products.

Features of the KLD enzyme mix used for Site-Directed Mutagenesis

• Fast : 5-15 min room temperature reaction.
• Useful : compatible for point mutation, 80 base insertions and unlimited-size deletions
• Economical : No need to purchase 5′ phosphorylated oligos
• Efficient : 90-95% mutant colonies using regular 25-cycle PCR or a 10-cycle Fast & Steep PCR.
• Upgradable : combine the KLD enzyme mix with Fast & Steep PCR and DMT Competent Cells.

Note : When using site-directed mutagenesis strategies that utilize whole vector amplification, to ensure that no extra mutations are introduced in the backbone of the vector, it is strongly recommended to perform subcloning of the sequenced insert into a vector that hasn’t been amplified by PCR. Alternatively, the whole vector can be sequenced.

Recommendation : Use the Fast & Steep WVA protocol for site-directed mutagenesis to limit the number of PCR cycles to a minimum whule keeping the number of doublings very high. The risk of introducing undesired mutations drops drastically by at least 100 000-fold.

Content of the KLD Mix for Back-to-Back Site-Directed Mutagenesis

• KLD Mix : 10 ul / 20 ul / 50 ul
• KLD Mix 5x buffer : 30 ul / 50 ul / 120 ul
• Control 4 kb Amplicon : 5 ul / 5 ul / 10 ul

Manual and datasheet

KLD mix datasheet 2017-05-23