Blunt/TA Ligase Master Mix will ligate these substrates:
dsDNA
Nicked DNA/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
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.
Blunt/TA Ligase Master Mix improves yields for ends that typically react slowly
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.
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: 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.
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.
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.
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.
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
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. DOI: 10.1186/2047-217X-3-22 PubMedID: 25386338
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. DOI: 10.1186/s12864-015-1519-z PubMedID: 25927464
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. DOI: 10.1093/nar/gkv773 PubMedID: 26240383
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
Yamagishi, J., et al. (2017) Serotyping dengue virus with isothermal amplification and a portable sequencer Sci Rep; 7(1), e3510. PubMedID: 28580104
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