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I am working on building a real time PCR machine. Is there any chemistry set that I can purchase online to:

  1. Identify if PCR amplification was successfully done.
  2. Test fluorescence dyes that could be used to validate if the fluorescence detector optics worked; i.e. something I could excite with a specific wavelength and monitor for fluorescence.

I am looking for something that is not dangerous and only serves the single purpose of validating the real time PCR machine.

Mods please advise if I need to move to Chemistry StackExchange

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    $\begingroup$ Are you asking for specific and proven techniques ? or information on something like a thermocycler? Other than that, I have no idea what a pcr "machine" is. $\endgroup$
    – Macedon93
    Jan 13, 2015 at 7:14
  • $\begingroup$ I am asking for two things 1) Something that will confirm that PCR occurred (ie color change after 30 cycles of PCR). 2) Something that will fluoresce at a specific frequency when excited by an excitation frequency. $\endgroup$
    – EasyOhm
    Jan 14, 2015 at 3:22
  • $\begingroup$ What level of accuracy do you expect for the identification of the PCR product? $\endgroup$
    – WYSIWYG
    Jul 7, 2016 at 10:35
  • $\begingroup$ If you're looking for a simple fluorophore just to make sure your detector is working, Auto parts stores carry fluorescent dyes to track oil leaks. Probably won't be useful for measuring amount of DNA though. $\endgroup$
    – user137
    Jul 8, 2016 at 10:09
  • $\begingroup$ I just remembered that I used to extract fluorescent dyes from highlighters by breaking them open and passing ethanol through the marker. That would be another easy source of dyes to test your detector. $\endgroup$
    – user137
    Jul 8, 2016 at 10:16

1 Answer 1

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There are two different ways to detect PCR product in real-time PCR: dyes which bind to DNA in general, such as SYBR Green I, or probes specific to your PCR product. There's a summary of the benefits of each here, with the following table:

table comparing SYBR and Taqman real-time chemistries

You can buy either online; they'll be fairly expensive but I can't think of an obvious way to get them cheaper if you're interested in high-confidence validation.

[edit] forgot to mention that you will also need to normalise the reporter signal against a reference dye such as ROX (https://www.thermofisher.com/order/catalog/product/12223012).

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