Possible Causes: Components

· Primer concentration is too low.

· Primer concentrations not balanced. Make sure primers are present in equal concentrations.

· New primers are required. Some primers are immune to optimization. See Primer Design.

· Nested primers are required. Reamplify dilutions (1:10 to 1:1000) of the first reaction using nested primers .

· Contaminated primer. See Rescuing Contaminated PCR

Primers and Wayward PCR Primers.

· Template concentration is too low. Use a higher concentration of template.

· Template concentration is too high. Excessive template can inhibit the reaction by binding all the primers .

· Template is degraded. Check the integrity of the template by electrophoresis after incubation.

· Template: Target sequence is not present in target DNA. Redesign your experiment or try other sources of target DNA.

· Mg++ concentration is too low. This may compromise enzyme activity so try increasing the concentration by 0.2 mM
increments.

· dNTP concentration is too low. Normally concentrations between 20 - 200 μM are optimal.

· dNTPs degraded. Keep nucleotides frozen in aliquots, thaw quickly and keep on ice once thawed. Avoid multiple
freeze/thaw cycles.

· pH of the reaction buffer is too high.

· Reaction mixture is incomplete or degraded. Do a control check with positive control DNA.

· Buffer isn't diluted enough. Add more water.Possible Causes: Conditions

· Denaturing temperature is suboptimal. Try extending the time and/or increasing the temperature of the initial denaturation
step prior to cycling (5 minutes at 95° C is standard).

· Annealing temperature is too high. Start at 10° C below calculated optimal annealing temperature.

· Suboptimal extension time. Increase by 1minute increments, especially for LA PCR.

· Inhibitors are present. See Enhancers & Inhibitors in this Guide.

· Enhancers needed. Some reactions may amplify only in the presence of additives. See Enhancers & Inhibitors in this
Guide.

· Mineral oil problem. The reaction must be overlaid with highquality, nuclease-free light mineral oil. Do not use autoclaved
mineral oil.

· Reaction tubes are contaminated. tubes eliminates contaminants that inhibit amplification. See also How to Reduce Contamination.

· Too few cycles. Try doing 10 additional cycles at a constan annealing temperature (i.e. 55° C) and recheck.

· Thermal cycler didn't cycle. Check to see if the thermal cycler was actually turned on.

· Thermal cycler was programmed incorrectly. Check to see if times and temperatures are correct.

· Thermal cycler temperatures are too low in some positions. Do a set of control reactions to determine if certain positions give
low yields .

· Thermal cycler top was left open.