HighEnd™ Repair Kit

Applications


  • Preparation of PCR products, sheared or nebulized DNA, restriction-digested DNA, cDNA for blunt-end ligation

  • Conversion of 5′- and/or 3′-protruding ends to 5′-phosphorylated blunt-ended ones

Benefits


  • Fast and simple blunting and phosphorylation of DNA at once

  • Universal - preparing any kind of DNA for blunt-end ligation

  • Premium reagents - reproducible results

Catalog. # Product Name Size Price Qty
HER0101 HighEnd™ Repair Kit 40 r of 25 µl
€129.50
HER0105 HighEnd™ Repair Kit 200 r of 25 µl
€539.50

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Description

HighEnd™ Repair Kit is a premium tool designed for rapid and highly efficient DNA end-repair before the ligation reactions. PCR products, sheared or nebulized DNA, restriction-digested DNA, and cDNA can be blunted/phosphorylated in a couple of minutes and are ready for an efficient blunt-end ligation and cloning.

The Kit includes HighEnd™ Repair Blend – an optimized mix of T4 DNA Polymerase and T4 Polynucleotide Kinase. The 5′→3′ polymerase and 3′→5′ exonuclease activities of T4 DNA Polymerase form the blunt-ended DNA. T4 Polynucleotide Kinase phosphorylates 5′ DNA ends. The resulting DNA is a high-quality blunt-ended substrate for T4 DNA Ligase.

Up to 1-5 microgram of the linear DNA can be blunted and phosphorylated in one 20 min reaction. After the thermal inactivation, the reaction mixture can be used for blunt-end ligations.

HighEnd™ Repair Kit is an ideal choice for preparing for ligations the PCR products obtained with high fidelity polymerases like ALLin™ HiFi DNA Polymerase (HLE0201).

Protocols

Print version of the protocol: Product Insert HighEnd™ Repair Kit
Important Notes
  • Check the integrity and the concentration of the DNA prior the reaction.
  • Always repurify PCR products before end-repair.
  • Thaw and keep reagents on ice. Mix all components well before use.
  • The optimal DNA amount in the reaction depends on the lengths of the DNA fragment. For example 1 µg of
  • 1 kb linear DNA has ~3 pmol ends, but 1 µg of 100 bp linear DNA has 10 times more substrate for blunting/phosphorylation; i.e. even 30 pmol DNA ends. Therefore, the shorter is the DNA fragment, the less of it shall be used in micrograms for end-repair or for later ligation reaction.
  • For high DNA amounts upscale the reaction accordingly. For example 5 µg of short 100 bp fragment can be end-repaired in 100 µl reaction using 2-5 µl of HighEnd™ Repair Blend.
 
Prepare a 25 µl reaction:
Linear DNA in TE buffer or water up to 1 µg (up to 30 pmol ends)
1 µg of 1 kb linear DNA has ~3 pmol ends
1 µg of 0.5 kb linear DNA has ~6 pmol ends
1 µg of 0.1 kb linear DNA has ~30 pmol ends
10X HighEnd™ Buffer 2.5 µl
1 mM dNTP Mix 2.5 µl
PCR Water up to 24 µl
HighEnd™ Repair Blend, 1 r/µl 1 µl (max 2 µl)
  • Mix well; incubate for 20 - 30 min at 25°C.
  • Inactivate enzymes at 75°C for 20 min and keep cooled in case the ligation is performed immediately or keep frozen in case the ligation is performed later.
  • Alternatively, re-purify the DNA using PCR clean-up spin column kit, elute in 25 µl of water or TE and keep frozen.
  • For subsequent ligation and cloning follow the recommendations for Rally™ Rapid Ligation Kit (RLK0101).

Specifications

CAT. SIZE COMPONENTS COMPOSITION
HER0101 40 r of 25 µl 40 µl – HighEnd™ Repair Blend, 1 r/µl
1.5 ml - 10X HighEnd™ Buffer
0.5 ml - 1 mM dNTP Mix

Enzyme blend storage buffer contains Tris, 50% glycerol and other components.

10X HighEnd™ Buffer contains Tris, NaCl, MgCl2, DTT, and other components.

dNTPs serve as building blocks for filling-in reaction and as phosphate donors for phosphorylation.
 
 HER0105 200 r of 25 µl 5 x 40 µl – HighEnd™ Repair Blend, 1 r/µl
2 x 1.5 ml - 10X HighEnd™ Buffer
3 x 0.5 ml - 1 mM dNTP Mix


Storage


In the dark at -20°C.

 

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