Why do I get smeared PCR products?

Please see the following factors that can contribute to unspecific, smeared PCR products, and suggestions how to avoid it:

• too much starting template

  Check the concentration of the starting template. Make serial dilutions of template nucleic acid from stock solutions. Perform PCR using these serial dilutions.

• carry-over contamination

  If the negative-control PCR (without template DNA) shows a PCR product or a smear, exchange all reagents. Use disposable pipet tips containing hydrophobic filters to minimize cross-contamination. Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis.

• enzyme concentration too high

  When using HotStarTaq or Taq DNA Polymerase, use 2.5 units per 100 µl reaction.

• too many PCR cycles

  Reduce the number of cycles in steps of 3 cycles.

• Mg2+ concentration not optimal

  Perform PCR with different final concentrations of Mg2+ from 1.5–5.0 mM (in 0.5 mM steps) using the 25 mM MgCl2 solution provided (see table below):

Final Mg2+ concentration in reaction (mM)	1.5	2.0	2.5	3.0	3.5	4.0	4.5	5.0
Required volume of 25 mM MgCl2 per reaction (ul)	0	2	4	6	8	10	12	14

 

• Primer concentration not optimal or primers degraded

  Repeat the PCR with different primer concentrations from 0.1–0.5 µM of each primer (in 0.1 µM steps). In particular, when performing highly sensitive PCR, check for possible degradation of the primers on a denaturing polyacrylamide gel.

• Primer design not optimal

  Review your primer design, and design new primers

For additional information on optimization of PCR results, please refer to the Appendix sections of the Taq PCR and HotStarTaq DNA Polymerase Handbook, and our comprehensive Brochure Critical Factors for Successful PCR.