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Home > Products > DNA Modifying Enzymes and Cloning > Cloning and Mutagenesis > Quick Blunting™ Kit > FAQ Quick Blunting™ Kit FAQ See the DNA Modifying Enzymes and Cloning FAQ also. Q1: Which ligase is recommended to ligate DNA treated with the Quick Blunting Kit? Q2: Can I use regular ligase to ligate my blunted product? Q3: I've blunted my DNA and now need to dephosphorylate the reaction with Antarctic Phosphatase. Do I need to purify the reaction? Q4: Does the PCR product need to be purified before blunting with the Quick Blunting Kit? What methods can be used to purify the product? Q5: I've digested my DNA and now want to blunt directly without purifying, can I add the blunting reagents directly to the restriction digest? Q6: I've blunted my sonicated gDNA for 15 minutes instead of the recommended 30 minute incubation time, will the reaction still work? Q7: I've accidentally skipped the heat-kill step after the blunting reaction. Will the ligation still work? Q1: Which ligase is recommended to ligate DNA treated with the Quick Blunting Kit? A1: The NEB Quick Ligation Kit (NEB #M2200) is recommended. Q2: Can I use regular ligase to ligate my blunted product? A2: Yes, but the ligation reaction should be incubated overnight at room temperature. A much lower transformation efficiency is seen with a shorter incubation time. Q3: I've blunted my DNA and now need to dephosphorylate the reaction with Antarctic Phosphatase. Do I need to purify the reaction? A3: 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. Q4: Does the PCR product need to be purified before blunting with the Quick Blunting Kit? What methods can be used to purify the product? A4: Yes, PCR reactions should be purified before blunting. We recommend Qiaquick PCR columns. Q5: I've digested my DNA and now want to blunt directly without purifying, can I add the blunting reagents directly to the restriction digest? A5: Yes, if the restriction enzyme is heat labile. Add 0.1 volume of dNTPs and 1 ul of Blunting Enzyme and incubate at room temperature for 15 minutes. If the restriction enzyme is not heat labile, the reaction will need to be purified using a commercial purifiction kit, phenol extraction/ethanol precipitation or gel electrophoresis. Q6: I've blunted my sonicated gDNA for 15 minutes instead of the recommended 30 minute incubation time, will the reaction still work? A6: 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. Q7: I've accidentally skipped the heat-kill step after the blunting reaction. Will the ligation still work? A7: Yes, but there may be a higher background since the polynucleotide kinase will still be active and can phosphorylate the vector. New England Biolabs, Inc. is an ISO 9001 and ISO 14001 Certified Company. NEB certifies that it is a small business in accordance with the US Small Business Administration and 13 CFR 121.201