Hi everybody,
I am developing a real-time PCR based animal MHC typing assay. I need to quickly decide whether SYBR Green or TaqMan chemistry should be used.
I am focusing on SYBR Green now (thought it's cheaper). I think about two, may three primers can be multiplexed with SYBR Green if their amplicon melting temperatures are sufficently different.
My assumptions are:
- Duplexing can be achieved with SYBR Green PCR;
- Multiplexing with up to 3 or 4 targets is possible with TaqMan;
- TaqMan master mix costs twice as much as SYBR Green master mix;
- Cost of synthesizing probes for TaqMan can be negligible if the assay will be used extensively in the future.
It seems like cost will be the same or TaqMan will be more expensive if these assumptions are true. But I am worried that multiplexing will not work with SYBR Green. In that case, is it safe to assume that the same primers can work with TaqMan (with probes developed additionally)? Can I proceed with SYBR Green for now and keep TaqMan as a backup plan?
Thanks Florian. The goal of the project is identification, not quantitation. Sounds like primers developed for SYBR Green approach can be used in TaqMan as well (probes aside). That's what I really need to know. Your answer helped a lot.
Just beware of the potential for dimers between the probe and primers. Also amplicon lengths for TaqMan should ideally be
Thanks you very much. We currently have old primers generating amplicons in 100-200 bp range, hope it will do.
Dear Priller,
Primer will work with Syber and Taqman both.
At the time of Taqman design select sequence within amplicon.
Taqman is specific and sensitive but costly compare to Syber green.
For multiplex go for Taqman chemistry using different lebeled Taqman probe which compitable to RTPCR machine.
Syber green also can use, interpretation based on melting cure analysis but it is tricky.
Best
Subash
Hi u can use any primers with probes just make sure that not cross binding to give dimmer.use multiple primer analyser to test that
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