Each lot of the TripleMaster PCR System is performance-tested in long range PCR. Routinely, TripleMaster PCR System is tested with genomic DNA using specific primers for the human tPA gene to obtain 24 and 27 kb products as well as with DNA and specific primers to amplify a 40 kb product.

8 Buffer choice

Choosing the appropriate reaction buffer is essential for obtaining optimal results with the TripleMaster PCR System. Use Table 1 as a guide for buffer selection in conjunction with the target size and template used. For targets ranging between 2 - 10 kb it is recommended to try both buffers and then choose the one with the best ratio of yield to specificity. For amplification of targets <= 500 bp or those with a low template concentration (< 10 ng/50 l of genomic DNA or < 500 pg/50 l of plasmid DNA) increase the final buffer concentration from 1x to 1.6x by adding 8 l of the 10x buffer to a 50 l assay. The resultant higher ionic strength of the reaction medium forces the TripleMaster Polymerase Mix to amplify short DNA fragments with a higher yield on small amounts of template DNA. Table 1: Buffer selection guide

9 Protocols

9.1 Long range PCR

Amplification with TripleMaster PCR System using the Tuning Buffer is robust and efficient for long targets up to 50 kb, even those where other enzymes fail.
General hints
- Use high molecular weight DNA templates only, stored at 28C. Avoid freezing the template DNA. Check the quality of the DNA; the average size must be > 50
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