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Support FAQs qPCR Assay Design & Optimization Why is my BHQ-Pulsar probe not detected in a singleplex reaction on the Lightcycler?
Support FAQs qPCR Assay Design & Optimization Why is my BHQ-Pulsar probe not detected in a singleplex reaction on the Lightcycler?
Why is my BHQ-Pulsar probe not detected in a singleplex reaction on the Lightcycler? |
If you are using a Lightcycler® 2.0 the most likely explanation is an instrumental issue. The Lightcycler instrument seeks FAM fluorescence from each capillary during the optics optimization procedure. In a singleplex reaction with only a BHQ-Pulsar 650 probe the instrument will not detect the capillary. To overcome this instrumental issue, FAM calibration dye needs to be spiked into any capillary that is lacking a FAM probe (Pulsar 650 singleplex reactions). We recommend spiking this dye to a final concentration of 10nM. A FAM calibration dye should have been included with your first order of BHQ-Pulsar probe. Please contact us if we can provide you with additional FAM calibration dye.
If you are using a Lightcycler® 1.2, which does not seek out FAM fluorescence during the optical optimization procedure. Simply raising the pre-set seeking temperature can resolve the detection of the BHQ-Pulsar Probe. Try raising the seek temperature between the ranges of 50° C to 95° C until an adequate signal is detected.
