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.