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DNA Blunting Kit

Features

  • Rapid blunting reaction
  • Excellent performance in blunt-ended ligation
  • Ready-to-use blunted product in subsequent ligation

Applications

  • Blunting and phosphorylation of double-stranded DNA
  • Preparation of restriction enzyme digested DNA or sheared DNA for blunt-ended ligation

Purchase DNA Blunting Kit

Availability: In stock Bulk requests please inquire at oem@biotechrabbit.com

Catalog # Size Package Content Price* Qty
BR1101101 100 rxn of 25 µl 125 µl 20× Blunting Enzyme Mix
250 µl 10× Bunting Buffer
25 µl 10 mM dNTP Mix
€298.00

*Does not include applicable tax, shipping & handling, or other charges.

OR

biotechrabbit™ DNA Blunting Kit shows excellent performance in blunting and phosphorylation of double-stranded DNA. The kit is compatible with a wide variety of dsDNA templates such as restriction enzyme digested DNA fragments, plasmid DNA, PCR products with dA-overhang, sheared or nebulized DNA. The kit is optimized for rapid blunting reaction.

 

 

Component

Composition

Blunting Enzyme Mix

20× Blunting Enzyme Mix, in storage buffer containing 50% (v/v) glycerol.

Blunting Buffer

Optimized 10× Blunting Buffer for rapid blunting reaction.

10 mM dNTP Mix

dNTP Mix containing 10 mM dATP, dCTP, dGTP, dTTP in water.

 

STORAGE

-20°C (until expiry date – see product label)

 

 

Quality Control
Functional Assay

Purified double digested sticky-end DNA is blunted, ligated and successful ligation is confirmed.

Exonuclease Activity

Linearized lambda/HindIII DNA fragments are incubated with the enzyme in a 50 µl reaction mixture for 4 h at 37°C. No DNA degradation observed.

Endonuclease/Nick Activity

Supercoiled plasmid DNA is incubated with the enzyme in a 50 µl reaction mixture for 4 h at 37°C. No conversion of covalently closed circular DNA to nicked DNA detected.

Contamination with E. coli DNA

Absence of E. coli genomic DNA is confirmed by qPCR using a sample of the enzyme and specific primers targeting the E. coli 16S rRNA gene. No contamination detected.

 

Blunting reaction
  • Scale the reaction volume up as required.
  • Prepare the blunting reaction as shown below:

 

Component

Volume

Final concentration

10× Blunting Buffer

2.5 µl

Purified DNA

Variable

up to 5 µg

10 mM dNTP Mix

0.25 µl

0.1 mM

20× Blunting Enzyme Mix

1.25 µl

Nuclease free water

Variable

 

Total volume

25 µl

 

 

  • Mix and centrifuge briefly to collect the liquid in the bottom of the tube.
  • Incubate at 25°C (room temperature) for 15 minutes.
  • For better efficiency or sheared/nebulized DNA extend the incubation time to 30 min.
  • Heat inactivate the enzyme at 70°C for 10 minutes.
  • Blunted DNA can be directly used in ligation step (overnight ligation suggested).

 

 

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