Hot Start method

This method is helpful for eliminating non-specific reactions with either TaKaRa Taq or

TaKaRa LA Taq. It suppresses adverse effects of the 3' to 5' exonuclease activity on the primers

An example of the Hot Start using TaKaRa LA Taq and wax beads (Mg free):

1. Add 2 of 10x LA PCR Buffer ll (Mg free), 2 of 25 mM MgCl, 8 of dNTP Mixture,

    0.5 each of the respective primers (10 pmol each) and 7 of dHO to bring the lower layer

    to a total of 20 .

2. After the careful addition of one wax bead on the lower layer, melt the bead at 70 for 5 minutes

    and then cool down to 10. Incubate for 5 minutes at 10.

3. Add 30 of the upper layer premix [containing 3 of 10x LA PCR Buffer ll (Mg free),

    3 of 25 mM MgCl, 0.5 of TaKaRa LA Taq (2.5 U), 0.5 of template DNA and 23

    of dHO] onto the melted wax.

4. Begin cycling normally.