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Immobilized T4 DNA Ligase

Catalog # Concentration Size List Price Quantity Your Price
M0569S 10 mg/ml 1.10 mg $464.00
$417.60
Catalog # Size List Price Your Price
M0569S 1.10 mg $464.00
$417.60
Catalog #
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*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

  M0569_IMT4_mechanism_0221

  • Immobilized T4 DNA Ligase (IM T4 DNA Ligase) consists of T4 DNA Ligase covalently bound to magnetic beads. 
  • Easily remove enzyme from reaction using a magnet 
  • Avoid heat inactivation step, which may interfere with downstream applications
  • Re-use immobilized enzymes 
  • Enable new workflows and applications
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    Learn about Immobilized T4 DNA Ligase

T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5´ phosphate and 3´ hydroxyl termini in duplex DNA or RNA. T4 DNA Ligase joins blunt end and cohesive end termini as well as repairs single stranded nicks in duplex DNA and some DNA/RNA hybrids (1).

Immobilized T4 DNA Ligase (IM T4 DNA Ligase) is a slurry of magnetic beads coated with T4 DNA Ligase to produce a 10 mg/ml solution (50% glycerol) with an effective concentration of 60 cohesive end units (CEU) per microliter of slurry. Following a reaction, enzyme can be removed using a magnet, and can be re-used.

 

Relative Activity of Immobilized T4 DNA Ligase at different temperatures

Temperature

% Activity

16ºC

100%

25ºC

100%

37ºC

50%

Activity of Immobilized T4 DNA Ligase in Different NEB Buffers

Buffer

pH

% Activity

NEBuffer r1.1

7.0

50%

NEBuffer r 2.1

7.9

100%

NEBuffer r3.1

7.9

70%

rCutSmart Buffer

7.9

100%

Phi29 DNA Polymerase Buffer

7.5

100%

Standard Taq Buffer

8.3

70%

Thermopol Buffer

8.8

70%

Q5 Reaction Buffer

8.5

10%

T4 DNA Ligase Buffer

7.5

100%

NEBNext Quick Ligation Buffer

7.6

25%

 


Figure 1: Successful ligation of Lambda DNA- HindIII Digest by Immobilized T4 DNA Ligase



Ligations of Lambda DNA- HindIII Digest (NEB #N3012) were performed with either no ligase, soluble T4 DNA Ligase (NEB #M0202) or Immobilized T4 DNA Ligase (NEB #M0569) according to recommended protocols.  Results indicate ligation is equivalent between soluble and immobilized enzyme.



Figure 2:  Immobilized T4 DNA Ligase can be re-used 



Consecutive splint ligations of Nanopore Adaptors were performed with Immobilized T4 DNA Ligase (NEB #M0569) in Quick Ligation Buffer. The data shows that activity remains unaffected after 20 consecutive ligation reactions. 

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  Immobilized T4 DNA Ligase M0569SVIAL -20 1 x 0.11 ml 10 mg/ml
  T4 DNA Ligase Reaction Buffer B0202SVIAL -20 1 x 1 ml 10 X
An exception occurred during the operation, making the result invalid. Check InnerException for exception details.

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Notes
  • ATP is an essential cofactor for the reaction. This contrasts with E. coli DNA ligase which requires NAD.
  • If IM T4 DNA Ligase is less than 10% of the total reaction volume, set-up reaction in the following order: H2O, buffer, DNA and finally enzyme.
  • If IM T4 DNA Ligase is more than 10% of the total reaction volume, we recommend setting up the reaction as follows:  Prepare the substrate in 1X reaction buffer.  Add IM T4 DNA Ligase into a fresh tube.  Put the tube on a magnet for 3 minutes to pellet the beads.  Then carefully remove the supernatant from the bead pellet and add the substrate mixture to the bead pellet. (Do not remove the supernatant until you are ready to assemble the reaction, as the beads should not be allowed to dry out.) 
  • If incubation is longer than 30 minutes, agitation will be required to ensure beads remain in suspension.
References
  • Engler, M.J. and Richardson, C.C. (1982). P.D. Boyer(Ed.), 5, 3. 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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