Causes

Trouble-shooting measures

  Concentration of primers is too high

Reduce the amount in decrements of 0.1 .

  Primes are not well designed for

Increase the specificity of the primers by

  the target sequence.

changing the complimentary region of the

template, or by elongating the primer length to

30 to 35 bases.

  Enzyme concentration is too high.

Reduce the amount in decrements of 0.5 U.

  Cycle number is too high.

Reduce the number of cycles in decrements of

2 cycles.

  Annealing temperature is too low.

Raise the temperature in increments of 2.

  Non-specific annealing of primers

Avoid this phenomenon through the use of

  occursduring heating from room

eithe Hot Start method using Wax beads

  temperature to denaturation

(Mg free) or Master Mix method. (improved

  temperature of 94 to 98.

resultsare as follow.)

  Extension time is too short.

Increase the extension time in increments of

1 minute.

  Denaturation is not complete.

Optimize denaturation conditions by

extending the time in increments of 5 seconds,

together with raising the temperature in

increments of 0.5

  Template concentration is too high.

Reduce the amount in decrements of 20% of

the previous one.