Standard LA PCR Protocol

Summarizing all the factors and parameters listed in the Chapter 2, here are standard

procedures which can be modified to meet individual research requirements.

1. Preparation of PCR reagent mixes

Method A (standard method)

*Overlay 50 of mineral oil, if necessary.  

Method B (Hot Start method with wax beads (Mg free))

Lower mix preparation  

Wax layer formation

Carefully add one wax bead (Mg free) to the tube. Melt the bead by heating the

reaction tube at 70 for 5 minutes. Then, cool down the tube at 20 for 2 minutes.  

Upper mix preparation

Carefully add 30 of the upper mix onto the wax layer, and begin cycling.

Method C (Master Mix method)  

Master Mix A preparation  

Master Mix B preparation

Place 25 of the Master Mix A to a tube on ice. Add 25 of Master Mix B and mix

   gently. Then, overlay 50 of mineral oil, if necessary.    

2. PCR conditions  

Apparatus and equipment

Authorized thermal cycler and 0.2 ml thin-wall type PCR reaction tubes  

(1) Simple cycling

(2) Autosegment extension**

*   When normal type of PCR tubes are used, denaturation time per cycle should be 20 seconds.  

**  Autosegment extension: At the 15th cycle and following, the extension time should be extended by 15 seconds

     each cycle, i.e. 15 minutes and 15 seconds at the 15th and 15 minutes and 4 minutes (19 minutes) at the

     30th cycle. This extension procedure may increase the yield of amplification products.