Dephosphorylation Tips:A clean-up step prior to addition of the phosphatase is not needed when trying to dephosphorylate a fragment after a restriction enzyme digest, if the restriction enzyme(s) used are heat inactivable. Alternatively, if the restriction enzyme(s) used are not heat inactivable, a DNA clean-up step is recommended prior to the dephosphorylation step.
When working with Shrimp Alkaline Phosphatase (rSAP, NEB #M0371) or Antarctic Phosphatase (NEB #M0289), which are heat-inactivatable enzymes, a DNA clean-up step after dephosphorylation is not necessary prior to the ligation step. However, when using Calf Intestinal Phosphatase (CIP, NEB #M0290), a clean-up step prior to ligation is necessary.
Antarctic Phosphatase requires the presence of Zn2+ in the reaction, so don’t forget to supplement the reaction with 1X Antarctic Phosphatase Reaction Buffer (NEB #B0289) when using other NEBuffers.
Phosphorylation Tips:The addition of PEG 8000 (up to 5%) can improve results.
T4 Polynucleotide Kinase (NEB #M0201) and T4 DNA Ligase (NEB #M0202) can be used together in the T4 DNA Ligase Buffer (NEB #B0202).
T4 Polynucleotide Kinase is inhibited by high levels of salt (50% inhibition by 150 mM NaCl), phosphate (50% inhibition by 7 mM phosphate) and ammonium ions (75% inhibited by 7 mM (NH4)2SO4).
If using T4 Polynucleotide Kinase and working with 5´-recessed ends, heat the reaction mixture for 10 min. at 70°C, chill rapidly on ice before adding the ATP (or Ligase buffer containing ATP) and enzyme, then incubate at 37°C.
T4 Polynucleotide Kinase requires DTT in the reaction. DTT in the buffer can be degraded over time following repeat freeze-thaws. If the reaction is not performing as expected, use fresh buffer (< 1 year) or add fresh DTT to 5 mM using a 1M stock.
Blunting Tips:Make sure that you choose the correct enzyme to blunt your fragment. The Quick Blunting™ Kit (NEB #E1201), T4 DNA Polymerase (NEB #M0203) and DNA Polymerase I, Large (Klenow) Fragment (NEB #M0210) will fill 5’ overhangs and degrade 3’ overhangs. Mung Bean Nuclease (NEB #M0250) will allow you to degrade 5’ overhangs.
When trying to blunt a fragment after a restriction enzyme digest, if the restriction enzyme(s) used are heat inactivable, a clean-up step prior to blunting is not needed. Alternatively, if the restriction enzyme(s) used are not heat inactivable, a DNA clean step is recommended prior to blunting.
When trying to blunt a fragment amplified by PCR, a DNA clean-up step is necessary prior to the blunting step to remove the nucleotides and polymerase.
When trying to dephosphorylate a fragment after the blunting step, you will need to add a DNA clean-up step after the blunting and before the addition of the phosphatase.
T4 DNA Polymerase and DNA Polymerase I, Large (Klenow) Fragment are active in all NEBuffers. Please remember to add dNTPs.
When trying to blunt a fragment with Mung Bean Nuclease, the recommended temperature of incubation is room temperature, since higher temperatures may cause sufficient breathing of the dsDNA ends that the enzyme may degrade some of the dsDNA sequence. The number of units to be used and time of incubation may be determined empirically to obtain best results.
Mung Bean Nuclease reactions should not be heat inactivated. Although Mung Bean Nuclease can be inactivated by heat, this is not recommended because the DNA begins to "breathe" before the Mung Bean Nuclease is inactivated, and undesirable degradation occurs at breathing sections. Purify DNA by phenol/chloroform extraction and ethanol precipitation or spin column purification.