Long and accurate PCR.

INTRODUCTION The following protocol illustrates the rules of successful long PCR: No more than 1 ng of template DNA is used per microliter of PCR in a 100-μl reaction; approximately 0.1 μl of KlentaqLA (not plain Taq) is used per kilobase of target (for targets >10 kb, 1-1.3 μl of enzyme should be used); the Mg++ concentration is considered as the excess over the level of dNTPs; a final concentration of 1.3 M betaine is included, and the denaturation temperature is 2-3oC lower than would be used without betaine; the reactions are preheated for 5 minutes at 69oC so that the 20-second window for denaturation will be achieved accurately during the first cycle; the duration of the denaturation steps has been minimized and does not exceed 10 seconds. This protocol has only one no-template control, with one of the primer pairs. For high numbers of cycles (25-40), it is also advisable to include a no-template and/or a one-primer control for each target.