New England Biolabs, E1201S, 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. Related Categories DNA Manipulation Applications Blunting,, Phosphorylation (Kinase),, PCR, FAQ Q: Which ligase is recommended to ligate DNA treated with the Quick Blunting Kit? A: The NEB Quick Ligation Kit NEB #M2200 is recommended. Q: Can I use regular ligase to ligate my blunted product? A: Yes, but the ligation reaction should be incubated overnight at room temperature. A much lower transformation efficiency is seen with a shorter incubation time. Q: I've blunted my DNA and now need to dephosphorylate the reaction with Antarctic Phosphatase. Do I need to purify the reaction? A: Yes, the reaction will need to be purified prior to dephosphorylation with Antarctic Phosphatase, CIP or any other commercially available phosphatase. The reaction can be purified using a commercial purification kit, phenol extraction/ethanol precipitation or gel electrophoresis. Q: Does the PCR product need to be purified before blunting with the Quick Blunting Kit? What methods can be used to purify the product? A: Yes, PCR reactions should be purified before blunting; we recommend the Monarch PCR & DNA Cleanup Kit. Q: I've digested my DNA and now want to blunt directly without purifying, can I add the blunting reagents directly to the restriction digest? A: We recommend to heat inactivate the restriction enzyme after a restriction digest and prior to the blunting reaction. If the restriction digest has been carried out with enzymes that can be inactivated by a heat treatment, then after the heat inactivation step, add 0.1 volume of dNTPs and 1 ul of Blunting Enzyme and incubate at room temperature for 15 minutes. If the restriction enzymes used are not inactivated by a heat treatment, then the reaction will need to be purified using a commercial purification kit (we recommend the Monarch PCR & DNA Cleanup Kit), or running the reaction on an agarose gel and then extracting the DNA (we recommend Monarch Gel Extraction Kit), or performing a phenol/chloroform extraction. Q: I've blunted my sonicated gDNA for 15 minutes instead of the recommended 30 minute incubation time, will the reaction still work? A: A slightly lower transformation efficiency is seen with shorter incubations but the reaction will still work. In general, 1.5 X fewer transformants are seen when PCR product or sheared/nebulized DNA is incubated for 15 minutes instead of 30 minutes. If a 15 minute incubation time is desired, increase blunting mix to 2 µl in the reaction. Q: I've accidentally skipped the heat-kill step after the blunting reaction. Will the ligation still work? A: Yes, but there may be a higher background since the polynucleotide kinase will still be active and can phosphorylate the vector.