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Hi-T4™ DNA Ligase

Catalog # Concentration Size List Price Quantity Your Price
M2622L 400000 units/ml 100000 units $422.00
$379.80
M2622S 400000 units/ml 20000 units $106.00
$95.40
Catalog # Size List Price Your Price
M2622L 100000 units $422.00
$379.80
M2622S 20000 units $106.00
$95.40
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

Hi-T4 DNA Ligase will ligate these substrates:

dsDNA




Nicked DNA/RNA





 

Featured Videos
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  • Monarch_MiniprepProtocol_VideoThumb
    DNA Ligation
  • Monarch_MiniprepProtocol_VideoThumb
    Quick Tips - Do I need a 5' phosphate for ligation?
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    NEBioCalculator® - Using the Ligation module
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    Golden Gate Assembly Domestication Tutorial
  • Monarch_MiniprepProtocol_VideoThumb
    Quick Tip - What is the ideal incubation temperature for ligation?
  • Monarch_MiniprepProtocol_VideoThumb
    Quick Tips - Troubleshooting problematic ligation reactions
  • Monarch_MiniprepProtocol_VideoThumb
    Quick Tips - How do I calculate how much DNA to add to a ligation reaction?
For details on NEB's quality controls for DNA ligases, visit our Ligase Quality page. 

A thermotolerant variant of T4 DNA Ligase, Hi-T4™ DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5´ phosphate and 3´ hydroxyl termini in duplex DNA or RNA and is designed to function at higher temperatures than wild type T4 DNA Ligase. Hi-T4 DNA Ligase will join blunt end and cohesive end termini as well as repair single-stranded nicks in duplex DNA, RNA or DNA/RNA hybrids at temperatures as high as 50ºC.

Figure 1: Hi-T4 DNA Ligase displays increased thermotolerance compared to T4 DNA Ligase. 


 
400 units of T4 DNA Ligase (NEB #M0202) or Hi-T4 DNA Ligase (NEB #M2622) were pre-incubated at 45°C in 1X T4 DNA Ligase Buffer in the absence of DNA substrate for the indicated time. Residual ligase activity was tested by adding 0.12 μM HindIII (cohesive end) fluorescently-labeled fragments and incubating at 37°C for 15 minutes. Ligation products were detected by capillary electrophoresis. 

T4 DNA Ligase steadily loses activity at 45°C, whereas Hi-T4 DNA Ligase retains full activity on a cohesive end substrate, even after 72 hours at 45°C. 


 
Figure 2: Hi-T4 DNA Ligase withstands higher temperature cycling than T4 DNA Ligase.





1,000 units of T4 DNA Ligase (NEB #M0202) or Hi-T4 DNA Ligase (NEB #M2622) in (1X) T4 DNA Ligase Buffer were cycled at 42°C (A) or 50°C (B) for 5 minutes followed by 16°C for 5 minutes in the presence of DNA (1 μg pBR322). Following the indicated number of cycles (0, 15, 30, 45 or 60), ligase activity was tested on a fluorescently-labeled, cohesive-end substrate (HindIII overhang, 25°C for 10 minutes). Ligation products were detected by capillary electrophoresis. Remaining ligase activity (relative to 0 cycle data point) is shown.  

A. T4 DNA Ligase gradually loses activity after cycling between 16°C and 42°C (gold line, A), while Hi-T4 DNA Ligase maintains activity after 60 cycles between 16°C and 42°C (orange line, A). 

B. T4 DNA Ligase is completely inactivated after only 15 cycles between 16°C and 50°C (gold line, B), while Hi-T4 DNA Ligase maintains ~60% activity after 60 cycles between 16°C and 50°C (orange line, B).

Highlights
  • Engineered thermotolerant variant of T4 DNA Ligase
  • Isolated from a recombinant source
Product Source
An E. coli strain that carries a plasmid encoding the engineered Hi-T4 DNA Ligase gene.
Features
  • Cloning of restriction fragments
  • Joining linkers and adapters to blunt-ended DNA
An exception occurred during the operation, making the result invalid. Check InnerException for exception details.

Companion Products
Materials Sold Separately
Notes
  • ATP is an essential cofactor for the reaction. This contrasts with E. coli DNA ligase which requires NAD.
  • To dilute Hi-T4 DNA Ligase that will subsequently be stored at -20°C, 50% glycerol storage buffer (Diluent Buffer A, NEB #B8001S) should be used; to dilute for immediate use, 1X T4 DNA Ligase Reaction Buffer can be used.

  • Ligation Temperature

    For cohesive (sticky) ends (1X T4 DNA Ligase Buffer), incubate 1 µl of Hi-T4 DNA Ligase in a 20 µl reaction between 25-50ºC for 10 minutes. For blunt ends or single base overhangs (1X StickTogether DNA Ligase Buffer), incubate 1 µl of Hi-T4 DNA Ligase in a 20 µl reaction between 25-50ºC for 10 minutes. Do not heat kill the reaction because heat treating the PEG in the StickTogether DNA Ligase Buffer will inhibit transformation.

References
  • Engler, M.J. and Richardson, C.C. (1982). P.D. Boyer(Ed.), 5, San Diego: Academic Press.
Quality Control Assay
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 Data Sheets
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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.

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