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Troubleshooting Guide for End Modification

 

Problem Cause Solution
Few or no transformants
 Inefficient phosphorylation
  • Purify the DNA prior to phosphorylation. Excess salt, phosphate or ammonium ions may inhibit the kinase.
  • If the ends are blunt or 5´ recessed, heat the substrate/buffer mixture for 10 minutes at 70°C. Rapidly chill on ice before adding the ATP and enzyme, then incubate at 37°C.
  • ATP was not added. Supplement the reaction with 1mM ATP, as it is required by T4 Polynucleotide Kinase (NEB #M0201).
  • Alternatively, use 1X T4 DNA Ligase Buffer (contains 1 mM ATP) instead of the 1X T4 PNK Buffer
Inefficient blunting
  • Heat inactivate or remove the restriction enzymes prior to blunting
  • Clean up the PCR fragment prior to blunting
  • Sonicated gDNA should be blunted for at least 30 minutes
  • Do not use > 1 unit of enzyme/μg of DNA
  • Do not incubate for > 15 minutes
  • Do not incubate at temperatures > 12°C (for T4 DNA Polymerase, NEB #M0203) or > 24°C (for Klenow, NEB #M0212)
  • Make sure to add a sufficient amount of dNTPs to the reaction (33 μM each dNTP for DNA Polymerase I, Large (Klenow) Fragment, NEB #M0210 and 100 μM each dNTP for T4 DNA Polymerase, NEB #M0203).
  • When using Mung Bean Nuclease (NEB #M0250), incubate the reaction at room temperature. Do not use > 1 unit of enzyme/μg DNA or incubate the reaction > 30 minutes.
Inefficient A-Tailing
  • Clean up the PCR prior to A-tailing. High-fidelity enzymes will remove any non-templated nucleotides