Preparation of primers and probes

You can basically use primers and probes available for the conventional real-time PCR insrument. As real-time PCR has been carried out in various researches all over the world, it is possible that you find appropriate sequences in the references.
For detaction of targets, various primers and probes are sold.*1

When you would like to use primeres and probes on the basis of sequence information already known, various manufacturers offer synthesis of them.
Please refer to examples of measurement as specific examples included.

Main points to considered when selectidng (or synthesizing) primeres and probes are as follows.
  • Recommended that the amplicon size is less than 150 bp.
  • You can consider Tm values for the PicoGene as the same meaning as for the conventional real-time PCR insrument. However, be sure that two primers(forward and reverse) have similar Tm values, and for the probe has several to 10℃ higher than for two primeres.
  • FAM dye, ROX™ dye, and Cy5™ dye could be used to Fluorescent dyes for the probes of PicoGene.
  • Be sure to add the FAM-labeled probe to the reagent for real-time PCR.
  • When performing multiplex, add primeres and probes related to ROX™ or Cy5™ in addition to the FAM. (PicoGene device supports up to 3 channels.)
  • When performing multiplex, please use dark quencher*2 as quencher for each probe.
  • When ordering primeres and probes, please request OPC grade for the primer and HPLC grade for the probe.
When purchasing sets of primer and probe , for example, the LightMix™ Series covers various targets.
LightMix™ is a registered trademark of Roche Molecular Systems, Inc.
When ordering the synthesis of probes, as a dark quencher, BHQ-1™ is often used for FAM, BHQ-2™ for ROX™, and BHQ-3™ for Cy5™.
Cy5™ is a registered trademark of GE Healthcare Bio-Sciences Corp.
BHQ™ is a registered trademark of Biosearch Technologies, Inc.
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