Product Class: Other

Blunt/TA Ligase Master Mix
Gray property icon for cloned at NEB recombinant Gray property icon for heat inactivation no

 Looking for additional T4 DNA Ligase formulations and variants?  Learn More

Product Introduction

Blunt/TA Ligase Master Mix will ligate these substrates:

dsDNA

dsDNA substrate for ligase selection:5´ [ straight line ] A-OH p [ straight line ] 3´3´ [ straight line ] p OH-T [ straight line ] 5´

dsDNA substrate for ligase selection:5´ [ straight line ] OH p [ straight line ] 3´3´ [ straight line ] p OH [ straight line ] 5´

dsDNA substrate for ligase selection:5´ [ straight line ] OH p [ straight line ] 3´3´ [ straight line ] p OH [ straight line ] 5´ 

Nicked DNA/RNA

Nicked substrate for ligase selection:5´ [ straight line ] OH p [ straight line ] 3´3´ [ straight line ] 5´

Nicked substrate for ligase selection:5´ [ wavy line ] OH p [ straight line ] 3´3´ [ straight line ] 5´


Ligase substrate key with [ black straight line ] DNA and [ red wavy line ] RNA 

 

 

Blunt/TA Ligase Master Mix is a ready-to-use solution of T4 DNA Ligase, proprietary ligation enhancer, and optimized reaction buffer. This master mix is specifically formulated to improve ligation and transformation of both blunt-end and single-base overhang substrates.

  • Simplifies reaction set-up, ensures an optimized ratio of enzyme and buffer components
  • No thawing is necessary as it remains liquid during storage at -20°C
  • Ligations for subcloning can be carried out in small volumes with low DNA concentrations, allowing users to conserve precious DNA samples and directly transform many strains of chemically competent E. coli without dilution
  • Suitable for Adaptor Ligation
  • Compatible with several Oxford Nanopore Technologies® workflows, including some involving Covid-19 research
  • Not sure which ligase to choose? Refer to our DNA and RNA Ligase Properties Chart or DNA Ligase Selection Chart

 

Bioz Badge Exists : True
Catalog # Size Concentration
M0367S 50 reactions 2 X
M0367L 250 reactions 2 X

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Product Information

Description

Blunt/TA Ligase Master Mix is a ready-to-use solution of T4 DNA Ligase, proprietary ligation enhancer, and optimized reaction buffer. This master mix is specifically formulated to improve ligation and transformation of both blunt-end and single-base overhang substrates. The master mix format simplifies reaction set-up, ensures an optimized ratio of enzyme and buffer components, and yields robust, rapid ligation of all types of DNA ends using a short incubation time at room temperature. No thawing is necessary as it remains liquid during storage at -20°C.* Ligations for subcloning can be carried out in small volumes with low DNA concentrations, allowing users to conserve precious DNA samples and directly transform many strains of chemically competent E. coli without dilution.

Learn more about Joining Difficult to Ligase dsDNA Fragments and Efficient Adaptor Ligation for the Preparation of dsDNA Libraries using Blunt/TA Ligase Master Mix.

Figure 1: Blunt/TA Ligase Master Mix improves yields for ends that typically react slowly

Bar graphs comparing yield on difficult-to-ligate ends
Yields of final ligation product for all reaction conditions using high concentration T4 DNA Ligase (NEB #M0202), the Quick Ligation Kit (NEB #M2200) and Blunt/TA Master Mix (NEB #M0367). Nick, cohesive end and 3´ single-base overhang substrates were incubated for 15 minutes; the 5´ single-base overhang was incubated for 1 hour.
* Freezers vary in their actual internal temperature. Our testing demonstrates that the master mix is liquid at -20°C. Freeze-thaw testing at -70°C has confirmed that the performance is unchanged after 20 freeze/thaw cycles.

Figure 2: Blunt/TA Ligase Master Mix works well at 25°C, and is complete in 15 minutes

Line graphs showing number of colonies versus time and temperature for Blunt/TA Ligase Master Mix
Ligation reactions with single-base overhang vector and insert were set-up using the Blunt/TA Ligase Master Mix and incubated for different times at 25°C (A) or at different temperatures for 15 minutes (B). Two microliters of each reaction were used to transform a 50 μl aliquot of NEB 10-beta Competent E. coli (NEB #C3019). Transformants resulting from triplicate plating 50 μl of a 1:5 dilution of the outgrowth were counted and graphed. The results indicate that the Blunt/TA Ligase Master Mix works well at 25°C, and is complete in 15 minutes.

Product Source

Purified from an E. coli strain containing a recombinant gene encoding T4 DNA Ligase.
This product is related to the following categories:
DNA Ligases
This product can be used in the following applications:
Cloning Ligation

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Heat Inactivation

No

Application Features

  • Vector construction
  • Linker ligation
  • Fragment assembly
  • Library construction
  • TA cloning

Product Notes

  1. DNA: Purified DNA for ligations can be dissolved in dH2O (Milli-Q® water or equivalent is preferable); TE or other dilute buffers also work well. For optimum ligation, the amount of vector DNA should be 20–100 ng and the insert should be added at a 3-fold molar excess. For ligation volumes greater than 10 μl, increase the volume of Blunt/TA Ligase Master Mix such that it remains 50% of the reaction. Insert:vector ratios between 2 and 6 are optimal for single insertions. Ratios below 2:1 result in lower ligation efficiency. Ratios above 6:1 promote multiple inserts. If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios.
  2. Time and Temperature: Most ligations performed using the Blunt/TA Ligase Master Mix reach an end point at 60 minutes or less when performed between 4–37°C. Incubation beyond this time provides no additional benefit. Our recommendation for a 25°C (room temperature) incubation was chosen after evaluation of performance at 4°C, 16°C, 25°C, and 37°C. Most conditions reached at least 50% performance within 15 minutes. Shorter times can also be used.
  3. Cells: Competent cells can vary by several logs in their competence. Perceived ligation efficiency directly correlates with the competence of the cells used for transformation. Always transform uncut vector as a control for comparison purposes.
  4. Electroporation: While electroporation can dramatically increase transformation efficiency, Blunt/TA Ligase Master Mix is not directly compatible with transformation by electroporation. It is necessary to reduce the PEG concentration. We recommend purification of the ligated DNA by spin column. 
  5. Biology: Some DNA sequences are not easy to clone. Sequences that form structures, including inverted and tandem repeats, are selected against by E. coli. Some recombinant proteins are not well tolerated by E. coli and can result in poor transformation or small colonies.

References

  1. Quick J., Quinlan, A.R., Loman N.J. (2014). A reference bacterial genome dataset generated on the MinION™ portable single-molecule nanopore sequencer. Gigascience. 3, 22. PubMedID: 25386338
  2. Madoui M.A., Engelen S., Cruaud C., Belser C., Bertrand L., Alberti A., Lemainque A., Wincker P., Aury J.M. (2015). Genome assembly using Nanopore-guided long and error-free DNA reads. BMC Genomics. 16, 327. PubMedID: 25927464
  3. Karamitros T., Magiorkinis G. (2015). A novel method for the multiplexed target enrichment of MinION next generation sequencing libraries using PCR-generated baits. Nucleic Acids Res.. 15, 43. PubMedID: 26240383
  4. Karlsson E., Lärkeryd A., Sjödin A., Forsman M., Stenberg P. (2015). Scaffolding of a bacterial genome using MinION nanopore sequencing. Sci Rep.. 5, 11996. PubMedID: 26149338

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. My transformations using Blunt/TA Ligase Master Mix reactions produced no colonies. What happened?
  2. Can the Blunt/TA Ligase Master Mix be used for the ligation of sticky-end fragments?
  3. Can the ligation reaction produced by the Blunt/TA Ligase Master Mix be directly used to transform electrocompetent cells?
  4. The recommended volume for a Blunt/TA Ligase Master Mix reaction is 10ul. I like to set-up my ligation reactions in a 20 ul volume. Can I scale-up the reaction?
  5. I routinely use more than 5 ul of my ligation reactions to transform 50 ul aliquots of competent cells. When I do this with the Blunt/TA Ligase Master Mix my transformation plates have very few colonies. What do you think is the problem?
  6. Can I incubate a Blunt/TA Ligase Master Mix ligation reaction for longer than 15 minutes?
  7. Can I incubate a Blunt/TA Ligase Master Mix ligation reaction at a temperature other than 25°C?
  8. My Blunt/TA Ligase Master Mix has frozen in my freezer. Is this a problem?
  9. The protocol for Blunt/TA Ligase Master Mix indicates the reaction should not be heat inactivated. How can I inactivate the ligation activity?

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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