Causes

Trouble-shooting measures

   Template concentration is too low.

Use sufficient concentrations. For a 50

PCR reaction, the amount of DNA should be

within the following ranges: Human genomic

DNA in a range of 0.1 to 1 g, E. coli

genomic DNA of 10 to 100 ng, and DNA

or plasmid DNA of 0.5 to 2.5 ng.

   Damaged template DNA was utilized.

Minimized damage in template DNA during

the preparation process. Avoid vortexing,

heat treatment, strong UV, shearing and ultra

sonication.

   Enzyme concentration is too low.

Increase the amount in increments of 0.5 U.

   Denaturation time is either too long or

Optimize the denaturation time in

   too short.

decrements or increments of 5 seconds.

   Denaturation temperature is either too

Optimize the temperature in decrements or

   high or too low.

increments of 0.5

   Annealing temperature is too high.

Lower the temperature in decrements of 2.

   Extension time is too short.

Lengthen the extension time in increments of

1 minute up to the optimum.

   Cycle number si too low.

Increase the number of cycles in increments

of 2 cycles up to the optimum.

   amplify the target sequence.

Design primers with high specificity to the

target DNA.