Product Class: Kit

Quick Blunting™ Kit

Product Introduction

The Quick Blunting™ Kit is used to convert DNA with incompatible 5´or 3´overhangs to 5´phosphorylated, blunt-ended DNA for efficient blunt-end ligation into DNA cloning vectors.

  • Restriction enzyme digested DNA is blunted in less than 30 minutes
  • Reactions are performed at room temperature in a ready-to-use mix
  • Suitable for restriction enzyme digested DNA, sheared or nebulized DNA or PCR product
Catalog # Size Concentration
E1201S 20.0 reactions
E1201L 100.0 reactions

Featured Videos

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  • DNABluntingVideo_thumb

    DNA Blunting Tutorial

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    The Mechanism of DNA Phosphorylation

Product Information

Description

The Quick Blunting Kit is used to convert DNA with incompatible 5´ or 3´ overhangs to 5´ phosphorylated, blunt-ended DNA for efficient blunt-end ligation into DNA cloning vectors. DNA is blunted using T4 DNA Polymerase (NEB #M0203) which has both 3´ → 5´ exonuclease activity and 5´ → 3´ polymerase activity. T4 Polynucleotide Kinase (NEB #M0201) is included in the enzyme mix for phosphorylation of the 5´ ends of blunt-ended DNA for subsequent ligation into a cloning vector. This kit is optimized for blunting up to 5 µg of DNA in a single reaction. To learn more about how to identify what type of overhang you have, visit this video tutorial.

Blunt Enzyme Mix supplied in: 100 mM KCl, 10 mM Tris-HCl(pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 0.1% Triton X-100 and 50% Glycerol.

1X Blunting Buffer:
100 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
0.025% Triton X-100
5 mM dithiothreitol
pH 7.5 at 25°C
This product is related to the following categories:
DNA Manipulation Products,
This product can be used in the following applications:
Blunting,
Phosphorylation (Kinase),
PCR,
Fast Cloning: Accelerate your cloning workflows with reagents from NEB

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E1201S     -20    
      Blunting Enzyme Mix M1201AVIAL -20 1 x 0.025 ml Not Applicable
      10X Blunting Buffer B1201SVIAL -20 1 x 0.5 ml 10 X
      Deoxynucleotide Solution Mix (1 mM) N1201AVIAL -20 1 x 0.1 ml 1 mM
  • E1201L     -20    
      Blunting Enzyme Mix M1201AAVIAL -20 1 x 0.125 ml Not Applicable
      10X Blunting Buffer B1201SVIAL -20 1 x 0.5 ml 10 X
      Deoxynucleotide Solution Mix (1 mM) N1201AAVIAL -20 1 x 0.5 ml 1 mM

Properties & Usage

Application Features

Prepare sheared, nebulized or restriction enzyme digested DNA for blunt-ended ligation into a plasmid, cosmid, fosmid or BAC vector

Prepare PCR products for efficient blunt-end cloning

Product Notes

  1. PCR generated DNA must be purified before blunting by using a commercial purification kit, such as Monarch® PCR & DNA Cleanup Kit (NEB #T1030), phenol extraction/ethanol precipitation, or gel electrophoresis.
  2. Restriction enzyme digested DNA can be blunted directly without purification.
  3. The Blunt Enzyme Mix has been optimized in Blunting Buffer, but is also active in NEBuffers 1,2,3 and 4, as well as BamHI, EcoRI and DpnII unique buffers when supplemented with dNTPs and dithiothreitol. There is a small reduction in ligation fidelity in these buffers. Transformation efficiency is lowest in NEBuffer 1 where the total yield is about 50% of optimum.
  4. ATP is not necessary for T4 Polynucleotide Kinase activity in the kit. The dATP and dTTP in the dNTP mix act as phosphate donors.
  5. The blunted reaction must be purified prior to phosphatase treatment by using a commercial purification kit, phenol extraction/ethanol precipitation, or gel electrophoresis.

Protocols, Manuals & Usage

Protocols

  1. Protocol for the Quick Blunting Kit (E1201)
  2. Transformation Protocol
  3. Quick Ligation Protocol (M2200)

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. Which ligase is recommended to ligate DNA treated with the Quick Blunting Kit?
  2. Can I use regular ligase to ligate my blunted product?
  3. I've blunted my DNA and now need to dephosphorylate the reaction with Antarctic Phosphatase. Do I need to purify the reaction?
  4. Does the PCR product need to be purified before blunting with the Quick Blunting Kit? What methods can be used to purify the product?
  5. I've digested my DNA and now want to blunt directly without purifying, can I add the blunting reagents directly to the restriction digest?
  6. I've blunted my sonicated gDNA for 15 minutes instead of the recommended 30 minute incubation time, will the reaction still work?
  7. I've accidentally skipped the heat-kill step after the blunting reaction. Will the ligation still work?

Troubleshooting

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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