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| 发布时间：2007-01-04|
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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.
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PCR反应抑制物