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.