End-Repair Mix

For converting DNA with 5′- and/or 3′-protruding ends to 5′-phosphorylated, blunt-ended DNA

S_1274_8_LS_OEM_Enzyme_EndRepair_MixLC200rx
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End-Repair Mix (low concentration)

Cat. No. / ID:  Y9140-LC-L

For 200 low-concentration DNA reactions. End-Repair Mix 1X (0.20 mL), 10X End-Repair Buffer (1 x 1.5 mL) and 1 mM dNTP solution (0.5 mL).
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$470.00
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Enzyme
End-Repair Mix (low concentration)
End-Repair Mix (high concentration)
The End-Repair Mix is intended for molecular biology applications. This product is neither intended for the diagnosis, prevention or treatment of a disease, nor has it been validated for such use either alone or in combination with other products.
Need bulk, customized or optimized products for commercial purposes? We also offer support with logistics, compliance and more. Reach out to cooperate with QIAGEN Strategic Partnerships & OEM

Features

  • Utilizes the 5′→3′ polymerase and 3′→5′ exonuclease activities of T4 DNA Polymerase
  • Includes T4 Polynucleotide Kinase to 5′-phosphorylate the ends of the blunt-ended DNA
  • Low-concentration formulation prepares ≤1 µg of DNA for blunt-end ligation
  • High-concentration formulation prepares >1 µg of DNA for blunt-end ligation
  • Blunted DNA is suitable immediately for ligation by T4 DNA Ligase

Product Details

The End-Repair Mix converts DNA containing damaged or incompatible 5′- and/or 3′-protruding ends to 5′-phosphorylated, blunt-ended DNA. The conversion to blunt-ended DNA is accomplished by exploiting the 5′→3′ polymerase and 3′→5′ exonuclease activities of T4 DNA Polymerase (P7080). T4 Polynucleotide Kinase (Y9040) ensures that the ends of the blunt-ended DNA fragments are 5′-phosphorylated for subsequent ligation by T4 DNA Ligase


Ligation may be performed immediately using Rapid T4 DNA Ligase (L6030-HC).


The low concentration End-Repair Mix formulation is optimized for procedures such as general cloning using low DNA concentrations.


The high concentration End-Repair Mix formulation is optimized for procedures such as library construction for next-generation sequencing using high DNA concentrations.


ATP is not required because the T4 Polynucleotide Kinase can utilize the deoxynucleotides (dATP and dTTP) used in the blunting reaction mix as phosphate donors.


The enzyme mixes are supplied in 100 mM KCl, 10 mM Tris-HCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 50% glycerol; pH 7.4 at 25°C.


The enzyme mixes are supplied with 1 mM dNTPs (N2060) and 10X End-Repair Buffer (B9140) containing 1 M Tris-HCl, 500 mM NaCl, 100 mM MgCl2, 50 mM DTT, 0.25% Triton X-100; pH 7.5 at 25°C.

 

Performance

Test Specification
Purity >99%
3’→5’ nuclease Functional
5’ phosphorylation Functional
5’→3’ DNA synthesis Functional
Double-stranded endonuclease 10 µL = No conversion
E. coli DNA contamination 10 µL <10 copies

 

Principle

The End-Repair Mix produces blunt-ended 5′-phosphorylated DNA by repairing DNA with damaged or incompatible 5′-protruding and/or 3′-protruding ends. The conversion to blunt-ended DNA is achieved by exploiting the 5′→3′ polymerase and 3′→5′ exonuclease activities of T4 DNA Polymerase (P7080). The action of T4 Polynucleotide Kinase (Y9040) in the mix ensures that the ends of the blunt-ended DNA fragments are 5′-phosphorylated for subsequent ligation. The end-repaired DNA can be efficiently and rapidly blunt-end ligated into plasmid, cosmid, fosmid, BAC, other cloning vectors, or next-generation DNA sequencing adaptors.

Procedure

Instructions for using End-Repair Mix (high and low concentration) are provided in the corresponding kit protocol in the resources below.

Quality Control


The purified enzymes in the End-Repair Mix (T4 DNA Polymerase P7040 and T4 Polynucleotide Kinase Y9040) are free of contaminating endonucleases. In addition, >99% enzyme purity was analyzed by SDS-PAGE. Negligible E. coli genomic DNA was confirmed by qPCR.


In a functional assay, 2 μL of End-Repair Mix was added to a double restriction enzyme digested, dephosphorylated plasmid DNA in 1X reaction buffer containing 0.1 mM dNTPs and incubated at 25°C for 30 minutes. Competent cells were transformed with the ligation mixture, plated onto LB/Amp/X-Gal plates and incubated overnight at 37°C. Control reactions consisting of End-Repair Mix without T4 DNA Polymerase and/or T4 Polynucleotide Kinase were tested in parallel. The efficiency of the reaction was evaluated by comparing the number of blue and white colonies present in the End-Repair Mix plates to those of the control plates.

 

Applications

These products are available for molecular biology applications such as:

  • Preparation of sheared or restriction enzyme digested genomic DNA for ligation of next-generation DNA sequencing adaptors
  • Preparation of double-stranded cDNA, produced from cellular RNA transcripts, for ligation of next-generation DNA sequencing adaptors
  • Preparation of sheared or digested DNA for blunt-end ligation into plasmid, cosmid, fosmid, or BAC vectors
  • Preparation of DNA amplified by PCR, containing A-overhangs, for efficient and cost-effective blunt-end cloning

 

Features End-repair Mix (low concentration) End-Repair Mix (high concentration)
Applications General cloning Library construction for next-generation sequencing
Sample amount ≤1 µg DNA >1 µg DNA
Number of reactions 200 75
Incubate at room temerapture Yes Yes
Heat inactivation (75°C for 20 minutes) Yes Yes
Supplied with reaction buffer Yes Yes
Supplied with dNTPs Yes Yes

Resources

Safety Data Sheets (1)
Certificates of Analysis (1)