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 | 1× |
Purified DNA | Variable | up to 5 µg |
10 mM dNTP Mix | 0.25 µl | 0.1 mM |
20× Blunting Enzyme Mix | 1.25 µl | 1× |
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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