( kb. - Use thin-walled 0.2 ml P...)TripleMaster PCR System

kb.
- Use thin-walled 0.2 ml PCR tubes.
- Ensure that the 0.2 ml PCR tubes fit exactly into the thermal block of the cycler. Eppendorf 0.2 ml PCR tubes are recommended for the Eppendorf Mastercycler.
- Always prepare two master mixes and chill them on ice.
- Set up the complete reactions on ice.
- Mix the final reaction by gently pipetting up and down (three times). Oil overlay is not necessary for cyclers equipped with heated lids.
- Always use an elongation temperature of 68C and a denaturation temperature of 93C.
- Use a final dNTP concentration of 500 M for targets > 10 kb.
- Primers should have annealing temperatures above 60C.
- Do not apply hot start techniques such as Ampliwax.

Protocol for long range 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 unspecific primer extension at lower temperatures.

1. Thaw the reagents listed in Table 2 carefully and keep on ice.
2. Make sure reagents are thoroughly thawed. Mix each reagent and briefly centrifuge before use.
3. If various amounts of the TripleMaster Polymerase Mix are to be tested, prepare an enzyme dilution in 1x Tuning Buffer as described in the Appendix.
4. Set up Mastermix 1 in a sterile microcentrifuge tube, mix well, centrifuge and place on ice (see Table 2).
5. Set up Mastermix 2 in a seperate sterile microcentrifuge tube, mix well, centrifuge and place on ice (see Table 2).

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