Quick Blunting™ Kit
The Quick Blunting™ Kit is used to convert DNA with incompatible 5´or 3´overhangs to 5´phosphorylated, blunt-ended DNA for efficient blunt-end ligation into DNA cloning vectors.
- Restriction enzyme digested DNA is blunted in less than 30 minutes
- Reactions are performed at room temperature in a ready-to-use mix
- Suitable for restriction enzyme digested DNA, sheared or nebulized DNA or PCR product
DNA Blunting Tutorial
The Mechanism of DNA Phosphorylation
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The Quick Blunting Kit is used to convert DNA with incompatible 5´ or 3´ overhangs to 5´ phosphorylated, blunt-ended DNA for efficient blunt-end ligation into DNA cloning vectors. DNA is blunted using T4 DNA Polymerase (NEB #M0203) which has both 3´ → 5´ exonuclease activity and 5´ → 3´ polymerase activity. T4 Polynucleotide Kinase (NEB #M0201) is included in the enzyme mix for phosphorylation of the 5´ ends of blunt-ended DNA for subsequent ligation into a cloning vector. This kit is optimized for blunting up to 5 µg of DNA in a single reaction. To learn more about how to identify what type of overhang you have, visit this video tutorial.
Blunt Enzyme Mix supplied in: 100 mM KCl, 10 mM Tris-HCl(pH 7.4), 0.1 mM EDTA, 1 mM dithiothreitol, 0.1% Triton X-100 and 50% Glycerol.
1X Blunting Buffer:
100 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
0.025% Triton X-100
5 mM dithiothreitol
pH 7.5 at 25°C
The following reagents are supplied with this product:
Store at (°C) Concentration 10X Blunting Buffer -20 10 X Blunting Enzyme Mix -20 20 reactions Deoxynucleotide Solution Mix (1 mM) -20 1 mM
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