Possible Causes: Components 可能原因之一——反应组分
· Primer concentration is too high.
· Primer degeneracy is too high.
· Nested primers are required.
· New primers are required. Some primers are immune to
optimization and it's possible that your primers are good
matches to other sites in addition to the desired template. See
Primer Design.
· Template denaturation efficiency is too low.
· Mg++ concentration is too high. Try decreasing the Mg++
concentration by 0.2 mM increments.
· dNTP concentration is too high.
· Polymerase concentration too high.
· pH is suboptimal. Check at higher and lower levels.

Possible Causes: Conditions 可能原因之二——反应条件
· Annealing temperature is too low, causing mispriming. Try
using a much higher annealing temperatures , especially in the
first few cycles.
· Cycles periods are too long.
· Too many cycles. Compare the number of bands and their
relative amounts after fewer cycles. There may be excessive
template if proportionally more of the intended product is
present at the earlier cycles.
· Ramp speed is too slow. Check your thermocycler
specifications.
· Inhibitors are present and/or concentrations are too high. See
About Inhibitors
· Enhancers are required. See About Enhancers.
· Contaminants are present. See How to Reduce Contamination.
· Hot Start or Touchdown PCR is required.