1. How does the FailSafe™ PCR System work?
The FailSafe PCR System is unique in that the optimal conditions for amplification are determined automatically by performing a single round of PCR, which is especially useful for solving tough PCR problems. The FailSafe™ PCR PreMix Selection Kit accomplishes this by using 12 different PCR PreMixes, which include buffer, deoxyribonucleotides, salt, and varying concentrations of magnesium salt and of the FailSafe™ PCR Enhancer (with betaine). The first time you use a specific template/primer combination, prepare PCR reactions using your template and primers with each of the 12 FailSafe™ PCR PreMixes. Analyze the PCR product from each PreMix reaction by agarose gel electrophoresis and visually determine which PreMix gives the best amplification: a single, strong PCR product of the desired length. Then use the selected PreMix for all subsequent amplifications with that template/primer combination. The product is called "FailSafe" because at least one of the 12 FailSafe PreMixes should give the desired product. Optimization is automatic. The time required for optimization of most PCRs is less than six hours, including DNA Purification, optimization and gel electrophoresis to confirm the best PCR PreMix to use in a reaction.
2. Can FailSafe be used for real-time (quantitative) PCR?
Yes. The FailSafe PCR System can be used "right out of the box" with fluorescent probes (TaqMan/Scorpion, etc) easily. For SYBR Green Real-Time PCR, it will be necessary for the end-user to add their own SYBR Green I Dye to the PreMixes at the appropriate concentration for the application to work. A separate tube of ROX dye must also be provided as a reference standard for use in real-time PCR instruments manufactured by Applied Biosystems. The optimization procedure for the FailSafe PCR Systems in Real-time experiments is identical to the standard FailSafe PCR System - simply run PCR using each individual Real-Time PCR PreMix and select the best PreMix based on the lowest threshold cycle (CT) and best melt curve.
3. What DNA Polymerase is used in the FailSafe System?
The FailSafe PCR Enzyme Mix is a proprietary blend of thermostable DNA Polymerases, and includes a proofreading polymerase. This combination of enzymes provides excellent PCR efficiency and much higher fidelity than systems that only use Taq DNA Polymerase, and allows amplification of even the toughest DNA templates. Regardless of the source or properties of the template, including those with high GC content, this enzyme mix efficiently amplifies the DNA.
4. Can I use Topo or TA-cloning with the PCR product from the FailSafe PCR System?
Yes. The FailSafe PCR System generates three different amplification product types, two of which are suitable for cloning. These product types are (1) PCR products with non-template A's on both ends and thus can be used in TA-cloning applications using T-vectors and also with Topo-TA cloning products; (2) PCR products that have blunt ends on both ends and thus can be blunt-end cloned into an appropriate vector; and (3) PCR products that have a non-template A on one end and a blunt end on the other. The ratios of the PCR product types will vary based on the template and no guidelines are available that will suggest PCR reaction types that will favor one product type over another.
5. Which FailSafe PreMix should I use for my GC-rich template? I don't want to test all of the PreMixes.
It is nearly impossible to determine, without experimentation, which FailSafe PCR PreMix will work best with a given template/primer combination. We strongly recommend using all of the PreMixes for optimization. If only selected PreMixes are tested, you run the risk of "missing" the optimal conditions for a given amplicons; and may end up selecting PCR conditions that may not be optimal for your reaction.
6. I want to amplify a template of about 20 kb. Should I use the FailSafe PCR System or the MasterAmp™ Extra-Long PCR Kit?
Both products can amplify templates around 20 kb. With FailSafe, for templates >10 kb, we recommend using 2.5 Units of the FailSafe PCR Enzyme Mix (for templates <10 kb, use 1.25 units in 50-μl reactions). For any template over 20 kb use the MasterAmp™ Extra-Long PCR System.
7. How good is the fidelity of the FailSafe PCR Polymerase enzyme mix?
The fidelity of the FailSafe enzyme mix is approximately three times better than Taq Polymerase; that is, the Taq Polymerase fidelity number is about one in 9000 bases; FailSafe's replication fidelity will be approximately one in 27,000 to 30,000 bases. While this may not be as good as other proofreaders (such as Pfu, Pwo or Phusion) it is easily sufficient for use in amplifying RNA-Seq or DNA-Seq libraries to be sequenced on any sequencing devise.
*Patents issued and pending.