2. The DNA template is too long (>5 kb).
3. The DNA template is GC rich and contains secondary structure. Use a polymerase better suited for high GC content, such as OneTaq® DNA Polymerase (NEB# M0480) or Q5® High-Fidelity DNA Polymerase (NEB# M0491). Alternatively, DMSO may be added to the reaction to lower the melting temperature of GC rich nucleic acids (1).
4. Primer concentration may be too low. We typically recommend 0.2 μM of each primer, but the range may be from 0.05-1 μM of each.
5. Try fresh nucleotides.
1. Sun, Y., Hegamyer, G. and Colburn, N. (1993). Biotechniques. 15, 372-374.
Traditional Cloning refers to the generation of DNA fragments using restriction enzymes, and their subsequent assembly into vectors and transformation. The name is derived from the method’s history as the first widely-accepted cloning method. Learn more in this tutorial about the benefits and disadvantages of Traditional Cloning.