Product Class: Other

KLD Enzyme Mix
recombinant 25

Product Introduction

  • Ability to phosphorylate and ligate in a single step
  • Degradation of template DNA by DpnI
  • Fast reaction time (5 minutes)
  • Combination of 3 enzymatic activities (kinase, ligase, DpnI) in a single enzyme mix
  • Also available as a kit: with and without competent cells
Catalog # Size Concentration
M0554S 25.0 reactions 10 X

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Product Information

Description

   

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).
This product is related to the following categories:
DNA Assembly, Cloning and Mutagenesis Kits Products
This product can be used in the following applications:
Site Directed Mutagenesis,
Site Directed Mutagenesis,
High-throughput cloning and automation solutions

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • M0554S     -20    
      KLD Enzyme Mix M0554SVIAL -20 1 x 0.025 ml 10 X
      KLD Reaction Buffer B0554AVIAL -20 1 x 0.15 ml 2 X

Properties & Usage

Application Features

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

Product Notes

  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.

Protocols, Manuals & Usage

Protocols

  1. KLD Enzyme Mix Reaction Protocol (M0554)

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  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?
  8. I use the Q5 Site-Directed Mutagenesis Kit to introduce single mutations. How can I introduce multiple mutations?

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

TroubleshootingTipsQ5SiteDirectedMutagenisis_720

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

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]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

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

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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