用Primer xpress软件来设计比较方便.docVIP

用Primer Express软件来设计比较方便、简单。网上搜索一下，我记得有网友提供的。如果实在找不到这个软件的话，可以参考下面的指南。先设计好Probe，再设计Primer。good luck。Design GuidelinesEnsure the following guidelines are met：? ?? ? × Amplicon length – 50 to 150 bases for optimum PCR efficiency.? ?? ? ×??Probe Length – 13 to 30 bases. Do not overlap primer and probe sequences.? ?? ? ×??Tm – 68 to 70 °C.? ?? ? ×??% GC – 30 to 80 %.? ?? ? ×??5 end – Cannot be a G residue. A G residue adjacent to the reporter dye will quench the reporter ? ?? ?? ?? ?fluorescence somewhat, even after cleavage.Avoid the following motifs：? ?? ? ×??Repeating oligonucleotides – Avoid runs of identical nucleotides. If repeats are present, there must be? ?? ?? ???fewer than four consecutive G residues.? ?? ? ×??Consecutive A residues – Avoid six consecutive A residues anywhere in the probe. Consecutive A ? ?? ?? ???residues can cause a high No Template Control (NTC) signal.? ?? ? ×??CC dinucleotides – Avoid two or more CC dinucleotides in the middle of the probe, which can sometimes? ?? ?? ???reduce signal. Select a different probe or design the probe using the anti-sense (complementary) strand.? ?? ? ×??FAM? dye-labeled probes – If you will order a FAM dye-labeled probe, avoid a G in the second position on the 5 end (VIC? dye-labeled probes are not affected ). A G in the second position on the 5 end in FAM dye-labeled probes can reduce fluorescent normalized reporter signal (Rn).? ?? ???×??Hairpin Loops, self-dimerization, and cross-dimerization.Design Considerations When Selecting Candidate Probes：? ?? ???×??Select probes with more C residues than G residues to minimize reporter fluorescence quenching. ? ?? ???×??Select probes with Tm 10 °C or more higher than the primer Tm. 定量PCR Taqman探针设计要领自90年代Taqman探针诞生以来，虽然荧光探针（引物）不断有新的技术出现，但是作为一种经典的定量PCR技术，Taqman探针技术仍然是许多实验研究人员进行定量检测的首选，这主要是因为相对于SYBR荧光染料，Taqman探针具有序列特异性，只结合到互补区，而且荧光信号与扩增的拷贝数具有一一对应的关系，因此特异性强灵敏度高，而且条件优化容易；而相对于杂交探针，Taqman探针只要设计一条探针，因此探针设计较便宜方便，而且也能完成基本的定量PCR要求。当然Taq