( e range for high fidelity PCR optimal ...)TripleMaster PCR System

e range for high fidelity PCR, optimal reaction parameters (incubation times, temperatures, concentrations of template DNA, primer, Mg2+ and enzyme mix) vary and must be determined for each primer/template pair individually.

Protocol for high fidelity PCR

Prepare two Mastermixes and keep them on ice. Mastermix 1 contains primers and template. Mastermix 2 contains enzyme, dNTPs and buffer. Combine Mastermixes 1 and 2 immediately prior to commencing cycling in order to avoid primer/template degradation by 35 exonuclease activity and non-specific primer extension at lower temperatures.

1. Thaw the reagents listed in Table 5 carefully and keep on ice.
2. Make sure reagents are thoroughly thawed. Mix each reagent and briefly centrifuge before use.
3. Prepare an enzyme dilution in 1x HighFidelity Buffer as described in the appendix, if variable amounts of enzyme units will be tested.
4. Set up Mastermix 1 in a sterile microcentrifuge tube, mix well, centrifuge and place on ice (see Table 5).
5. Set up Mastermix 2 in a seperate sterile microcentrifuge tube, mix well, centrifuge and place on ice (see Table 5).
6. Just before cycling, pipette Mastermix 1 and Mastermix 2 into a separate PCR tube on ice. Mix thoroughly (vortex) and centrifuge.
7. Place immediately into a thermal cycler pre-heated at 94C and start cycling. Example of cycling profile is given for the Eppendorf Mastercycler (see Table 6).
8. Analyze amplification products on a 0.61.5% agarose gel using appropriate DNA markers.

Table 5: Mastermix composition for high fidelity PCR
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