Hieff™ Versatile T4 DNA Ligase (600 U/µL)- Thermotolerant and High Fidelity 12996ES

SKU: 12996ES60

Size: 60 KU
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Description

Hieff™ Versatility T4 DNA Ligase is a variant of T4 DNA Ligase. This enzyme is suitable for catalyzing the ligation reaction between cohesive or blunt-ended molecules. It is a single-enzyme product designed for connecting DNA fragments and adapters during the library preparation process in next-generation sequencing(NGS). The Hieff® Versatility T4 DNA Ligase has been extensively validated through high-throughput sequencing and exhibits exceptional quality.

Feature

  • High fidelity
  • Thermotolerant

Learn more about the versatile T4 DNA ligase development

Specifications

Source Recombinant E.coli
Storage Buffer 50 mM Tris-HC1,100 mM KC1,0.1 mM EDTA,1 mM DTT,50%Glycerol,pH 7.50.2 @25℃
Unit Definition In a ligation reaction system of 20 μL, the enzyme amount required to catalyze the ligation of more than 50% of DNA fragments, when 6 μg of λDNA-Hind III is reacted at 16°C for 30 minutes, is defined as 1 unit of activity (U).

Components

Components Name

12996ES60 

60KU 

12996ES62

600KU 

12996-A Hieff® Versatile T4 DNA Ligase (600 U/µL) 100 μL 1000 μL
12996-B 10×T4 DNA Ligase Buffer 500 μL 3×1500 μL

Storage

This product should be stored at -25~-15℃ for 2 years.

Figure

Figure 1. Illustrates the evolution of Versatility T4 DNA Ligase. (A) Rational design  of T4 DNA ligase. (B) Screening of thermotolerant T4 DNA ligase mutants at 45℃, leading to the identification of mutants with significantly enhanced specific activity. (C) Screening of T4 DNA ligase mutants aimed at reducing adapter dimer formation during DNA library preparation.  (D) The evaluation of library yields retention post heat treatment at 42℃ for 1 hour using DNA library preparation method.


Table 1. Yeasen Versatile T4 DNA ligase is thermostable. The enzyme was treated at 30℃ for 5 days and analyzed by DNA Lib Prep method.

Figure 2. Yeasen Versatile T4 DNA ligase has higher fidelity indicated by less adapter dimer formation due to self mis-ligation (A) and analyzed by DNA Lib Prep method (B).

Figure 3. Yeasen Versatile T4 DNA ligase has higher fidelity indicated by less adapter dimer formation compared with other brand T4 ligases analyzed by DNA Lib Prep method.

Figure 4. The Versatile T4 DNA Ligase reduces false positive rates for FFPE samples. Using DNA Lib Prep kit, which includes the Versatile T4 DNA Ligase, we prepared libraries for three types of standard samples (FFPE, gDNA, ctDNA) and conducted a head-to-head comparison with supplier W's product. The data indicates that for both gDNA and ctDNA, the Soft Clip and false chimeric rates of 12996 are comparable to those of competitor W. However, 12996 shows a marked advantage in FFPE samples.


Please leave a message to inquire about the performance comparison data between this enzyme and other top-tier thermostable and conventional T4 DNA ligases.


Notes

1. Please operate with lab coats and disposable glovesfor your safety.

2. If a small amount of precipitation occurs in the buffer, it is a normal phenomenon. Please invert and mix the buffer thoroughly before using it.

3. This product is for research use only.

Protocol

1. The current mainstream DNA library preparation kits using the ligation-based method typically do not require purification steps after end repair and A-tailing. The DNA fragments are directly subjected to adapter ligation without purification. This kit is compatible with the mainstream commercially available end repair and A-tailing systems for seamless adapter ligation.

Set up the following reaction in a microcentrifuge tube on ice, gently mix by pipetting or shaking, Centrifuge briefly and heat inactivate at 20°C for 15 minutes.

 

Table 1. Reaction System for Adapter Ligation

Component

VolumeμL

Final concentration

dA-tailed DNA

60

10×T4 DNA Ligase Buffer

10

0.1 μM-0.5 μM

50% PEG6000

12*

1 ng -400 ng

DNA Adapter

5**

 

Hieff® Versatile T4 DNA Ligase (600 U/µL)

3~5***

 

Nuclease-free water

up to 100

-

Note*The 50% PEG6000 solution is not provided in the kit, you will need to prepare it yourself

**Please refer to Table 2 for the amount of adapter.

***The amount of T4 DNA Ligase can be added as needed by 3-5 μL.

2. The concentration of the adapter directly affects the ligation efficiency and library yield. Excessive use of adapters may result in more adapter dimers, while low usage may affect connection efficiency and library production. Table 2 lists the recommended adapter amount for different amounts of input DNA.

 

Table 2. The recommended adapter amount for 100 pg-1 μg Input DNA

Input DNA

Concentration

0.1 ng

0.1 μM

1 ng

0.5 μM

10 ng

1 μM

25 ng

2 μM

100 ng~1000 ng

10 μM

Note】:The amount of adpter can be adjusted according to the requirements in the table above.

 

 

 

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