New England Biolabs, M0554S, KLD Enzyme Mix

Related Categories DNA Assembly, Cloning and Mutagenesis Kits Applications Site Directed Mutagenesis,, Site Directed Mutagenesis,, High-throughput cloning and automation solutions Specification Materials Required but not Supplied Nuclease-free Water (NEB #B1500) FAQ Q: Is the KLD Mix (NEB#M0554) the same product as the component of the Q5 Site-Directed Mutagenesis Kits (E0554S and E0552S)? A: Yes. The KLD mix is the same product as the KLD component in the Q5 Site-Directed Mutagenesis Kits. Q: What polymerases can be used to amplify the fragments that will be phosphorylated and ligated by the KLD mix? A: The KLD mix is to be used with fragments that have been amplified with Q5 High Fidelity polymerase or Phusion High Fidelity polymerase. This product is not suitable for amplicons with 3´ overhangs (ex: Taq amplicons). We do not recommend the use of this product on DNA fragments that have been amplified by other polymerases. Q: Do I need to purify my PCR-amplified fragments before or after treatment with the KLD mix? A: No. Purification of the PCR-amplified fragments is not necessary. Q: What is the KLD Mix? A: The KLD Mix contains a blend of kinase, ligase and DpnI enzymes in a buffer that preserves the activity of the enzymes. This formulation allows efficient phosphorylation, intramolecular ligation/circularization and template removal in a single 5 minute reaction step at room temperature. Q: If I double my PCR size, should I add more PCR mix to the KLD reaction? A: No. The 10X KLD Enzyme Mix is formulated to work well with 1 μl of the PCR mix regardless of the volume of the PCR. Q: Can I use more than 5μl of the KLD reaction to transform E coli? A: No. If more than 5μl of the KLD reaction is needed, a buffer exchange step, such as PCR purification, should be included prior to transformation. Q: Can the KLD reaction be incubated longer or at higher temperature? A: The KLD reaction can be incubated for up to 60 minutes at room temperature. Alternatively, to increase efficiency of the DpnI digestion, the KLD reaction can be incubated at room temperature for 5 minutes, followed by 30-60 minutes at 37C. The room temperature incubation is essential for the activity of the kinase and ligase enzymes. Q: I use the Q5 Site-Directed Mutagenesis Kit to introduce single mutations. How can I introduce multiple mutations? A: NEB® has developed a protocol using NEBuilder HiFi DNA Assembly Master Mix to simplify the construction of multiple site-directed mutagenesis. The technique involves the design of complimentary flanking primers to align fragments. This application note describes the use of the NEBuilder HiFi DNA Assembly Master Mix to generate multiple site-directed mutagenesis at the same time. We recommend 18-20nt overlaps but the length of the primers can vary. The primers are completely overlapping as illustrated below. The goal is to achieve balanced Ta’s for the primer pairs. The mutations are positioned in the middle of the primer. There must be enough bases on either side, and most importantly 3’ of the mutation, so that the primer will anneal properly to the template.