KLD Enzyme Mix



KLD Enzyme Mix is a unique blend of Kinase, Ligase and DpnI enzymes. This formulation allows efficient phosphorylation, intramolecular ligation/circularization and template removal in a single 5 minute reaction step at room temperature. This master mix is a component of the Q5 Site-Directed Mutagenesis Kits and it has been designed for use with fragments that have been PCR-amplified by Q5 Hot Start High-Fidelity DNA Polymerase.

Q5 Site-Directed Mutagenesis Kit Overview
This kit is designed for rapid and efficient incorporation of insertions, deletions and substitutions into double-stranded plasmid DNA. The first step is an exponential amplification using standard primers and a master mix formulation of Q5 Hot Start High-Fidelity DNA Polymerase. The second step involves incubation with a unique enzyme mix containing a kinase, a ligase and DpnI. Together, these enzymes allow for rapid circularization of the PCR product and removal of the template DNA. The last step is a high-efficiency transformation into chemically competent cells (provided).

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
KLD Reaction Buffer-202X

Advantages and Features


  • Ability to phosphorylate and ligate in a single step
  • Degradation of template DNA by DpnI


  • Fast reaction time (5 minutes)
  • Combination of three enzymatic activities (Kinase, Ligase, DpnI) in a single enzyme mix

Properties and Usage

Storage Temperature



  1. This product has been designed to be used on dsDNA fragments that have been amplified by Q5 High-Fidelity Polymerase specifically.
  2. This product is to serve as a companion product for our Q5 Site-Directed Mutagenesis Kits.
  3. For best results, primers should be designed and annealing temperatures calculated using NEBaseChanger, the NEB online primer design software.


  1. Is the KLD Mix (NEB#M0554) the same product as the component of the Q5 Site-Directed Mutagenesis Kits (E0554S and E0552S)?
  2. What polymerases can be used to amplify the fragments that will be phosphorylated and ligated by the KLD mix?
  3. Do I need to purify my PCR-amplified fragments before or after treatment with the KLD mix?
  4. What is the KLD Mix?
  5. If I double my PCR size, should I add more PCR mix to the KLD reaction?
  6. Can I use more than 5μl of the KLD reaction to transform E coli?
  7. Can the KLD reaction be incubated longer or at higher temperature?

Tech Tips

Only use 5 μl of the KLD reaction in the transformation. If more KLD reaction is added, a buffer exchange step, such as PCR purification, should be included prior to transformation.

Only use 1 μl of PCR product in the KLD reaction. Carrying too much PCR product forward can decrease transformation efficiency. If the PCR yield is low, more product can be added to the KLD reaction, however a buffer exchange step, such as PCR purification, must be included prior to transformation.

Use NEBaseChanger™ to design primers and calculate annealing temperature

Troubleshooting tips for Q5 Site-Directed Mutagenesis Kit


  1. KLD Enzyme Mix Reaction Protocol (M0554)

Interactive Tools


The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.