What are the tips for amplification with In-Fusion PCR primers?
What are the tips for amplification with In-Fusion PCR primers?
-The template-specific 3' end of the In-Fusion PCR primer should be 18–25 nt long, in order to ensure template amplification.
-To determine the Tm of In-Fusion PCR primers, use independent software for PCR primer analysis, such as OligoAnalyzer 3.1 from IDT Technologies. Use standard Mg2+, Na+, and dNTP concentrations usually recommended for PCR.
-For optimal amplification, perform the initial 3–5 PCR cycles using the annealing temperature compatible with just the 3' template-specific portion of the In-Fusion PCR primer. The remaining PCR cycles should use an annealing temperature compatible with the Tm of the entire primer.
-If you experience inefficient PCR amplification, it may be necessary to re-design the In-Fusion PCR primers, by either extending or repositioning the template-specific 3' end.