Q4.  WHAT ARE THE RECOMMENDED CYCLING CONDITIONS FOR LA PCR?

Optimal conditions for LA PCR depend on the lengths of amplified DNA fragments,

reaction volume, thermal cyclers used, etc.

1. Cycle number: Set the optimum cycle number at 25 to 30 cycles.

2. Denaturation conditions: Select denaturation conditions by the instrument and reaction

    tubes to be used. For example, when using 0.2 ml thin-wall type tubes, either

    98 for 10 seconds or 94 for 20 seconds is optimal.

3. Annealing and extension conditions: For shuttle (or 2 step) PCR, determine the optimum     annealing/extension temperature by varying the temperature in 2 increments in the

    range of 60 to 68. When the anneal-extension temperature is fixed at 68, set the

    cycle time for 30 seconds to 1 minute per 1 kbp. In the case of a temperature below

    68, this time should be longer, as it is not optimal for this enzyme.

Annealing and extension conditions may be more finely optimized by utilizing

autosegment extension. Shown below is the result of autosegment extension carried out

with the LA PCR Kit Ver. 2.1.

Template:
Human genomic DNA
500 ng/50 PCR

-globin cluster region (21.5 kb)

Primer length:

35 mer