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T4 DNA Ligase

Ordering Information
Cat No Size
27013

100 §¡ (5 U/§¡)

27012

100 §¡ (1 U/§¡)



Price 27013

212.00 €

Add to Cart



Price 27012

97.00 €

Add to Cart

PROTOCOL


Description

Catalyzes the formation of a phosphodiester bond between juxtaposed 5¡¯phosphate and 3¡¯ hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt-end and cohesive-end termini as well as repair single strand nicks in duplex DNA, RNA or DNA/RNA hybrids.

Applications

  • General cloning
  • Sticky-ended ligation
  • Blunt-ended ligation
  • Linker/Adator ligation

Characteristics

Content

For Research use. T4 DNA ligase contains sufficient reagents to perform approximately 100 X 20§¡ DNA ligase reactions.

Material Provided

Quantity

27012

27013

T4 DNA ligase

100§¡(1U/§¡)

100§¡()5U/§¡)

T4 DNA ligase Reaction Buffer (10x)

200§¡

1ml

Unit Definition

One unit is defined as the amount of enzyme required to give 50% ligation of Hind III fragments of Lambda DNA (5¡¯ DNA termini concentration of 0.12uM, 300ug/ml) in a total reactive volume of 20§¡ in 30 minutes at 16¡ÆC in 1x T4 DNA ligase reaction buffer.

Quality Control

  • Purified free of contaminating endonucleases and exonucleases.
    Each lot is functionally tested in ligation reaction. Additionally, each lot is analyzed by SDS polyacrylamide gel electrophoresis for the presence of detectable (less than 5%) polluting additional proteins.
  • Endonuclease Activity :
    Incubation of a 50ul reaction containing 20 units of T4 DNA ligase with 1ug of supercoiled plasmid DNA for 4 hours at 37°C resulted in < 10% conversion to nicked or cut plasmid DNA as determined by agarose gel electrophoresis.
  • Physical Purity :
    The purity is ¡Ã 95% as judged by SDS-polyacrylamide gels with coomassie blue staining. T4 DNA ligase is free from intact DNA ligase and small fragment as indicated by SDS-polyacrylamide gel electrophoresis.

Storage & Stability

All component should be stored at -20°C.

The reaction buffer have been optimized for the T4 DNA ligase protocols. Do not substitute with buffers provided with other iNtRON kits.

Inactivation Condition

By heating at 65°C for 10min.

Reaction & Storage Buffer

Reaction Buffers (1x) Storage Buffers
30 mM Tris-HCl (pH 7.5)
10 mM Tris-HCl (pH 7.5)
0.1 mM ATP 50 mM KCl
10 mM MgCl2 1 mM Dithiothreitol
10 mM Dithiothreitol 50% glycerol

Protocol

We recommend using a 1:1, 1:3 molar ratio of vector:insert DNA when cloning an insert into a plasmid vector. These ratios will vary with other types of vectors, for example cDNA and genomic cloning vectors. The following example illustrates the conversion of molar ratios to mass ratios for a 4.0kb plasmid and a 0.5 kb insert DNA fragment.

(ng of vector) x (kb size of insert)
insert
x molar ratio of =ng of insert
kb size of vector
vector

[Example]
How much 0.5kb insert DNA should be added to a ligation in which 100ng of 6 kb vector will be used? The desired vector:insert ratio will be 1:3.

(100ng vector) x (0.5kb size of insert)
3
x molar ratio of = 250 ng of insert
6 kb size of vector 1
  1. Assemble the following reaction in a sterile microcentrifuge tube:
    vector DNA    100ng
    insert DNA    25ng
    Ligase 10X Buffer   2§¡
    T4 DNA ligase    1U
    Nuclease-Free Water to final volume 20§¡
  2. Incubate the reaction at:
    room temperature for 4 hour, or 4¡É overnight, or 15¡É for 4-18hours

Experimental Information
1. Physical Purity

Figure 1. Purity test of T4 DNA ligase
Lane M, Marker;
lane 1, T4 DNA ligase (Cat. No. 27012);
lane 2, T4 DNA ligase (Cat. 27013) Physical purity ¡Ã 95%

 

2. Functional Activity
1) Sticky end

 

Figure 2. Ligation of Hind III fragments (4-base overhang of lambda DNA using 1 unit of T4 DNA Ligase.) Incubation at 25¡É.

2) Blunt end

Figure 3. Ligation of blunt-ended Dra I fragments of Lambda DNA using various amounts of T4 DNA ligase in a 20 ul reaction volume. Incubation at 25¡É for 30 min.

 


 

  • Gene origin: T4 phage
  • Molecular Weight: 58.5 kDa
  • Preparation: purified by anion exchange chromatography
  • Physical Purity: ¡Ã 95% as judged by SDS-polyacrylamide gels with Coomassie blue staining
  • Source: a recombinant enzyme of the E.coli strain expressing a recombinant clone.
  •  

    T4 DNA Ligase (5 U/ul)
    Ordering Information | Cat No 27013| Size 100 §i (5 U/§i)
    212.00 €
    Add to Cart

     

    T4 DNA Ligase (1 U/ul)
    Ordering Information | Cat No 27012| Size 100 §i (1 U/§i)
    97.00 €
    Add to Cart

     
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