| Observation |
Recommended Solution(s) |
| No or low PCR yield |
For difficult templates (AT and GC rich). Try 2x PolyMate to lower the melting profile and improve performance. |
| Enzyme concentration too low – increase the amount enzyme in 0.5U increments. |
| Magnesium concentration too low – increase concentration in 0.25mM increments with a starting concentration of 1.75mM. |
| Primer concentration not optimised. Titrate primer concentration (0.3-1µM); ensuring that both primers have the same concentration. |
| Template concentration too low – Increase Concentration of template |
| Perform a positive control to ensure that the enzyme, dNTPs and buffers are not degraded and/or contaminated |
| Multiple Bands |
Primer annealing temperature too low. Increase annealing temperature. Primer annealing should be at least 5°C below the calculated Tm of primers. |
| Prepare master mixes on ice or use a heat-activated polymerase. |
| For problems with low specificity. Try Hi-Spec Additive or 2x PolyMate (not supplied) to improve specificity. |
| Smearing or artifacts |
Template concentration too high. Prepare serial dilutions of template. |
| Too many cycles. Reduce the cycle number by 3-5 to remove non-specific bands. |
| Enzyme concentration too high - decrease the amount of enzyme in 0.5U increments. |
| Extension time too long. Reduce extension time in 0.5-1 minute increments. |
