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.

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:

• 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).

Product manual

Other documentation