Why is my BHQ-Pulsar 650 probe not detected in a singleplex reaction on my LightCycler?
The Lightcycler 2.0 instrument 'seeks' detection of FAM fluorescence in each capillary during the optics optimisation procedure. In a singleplex reaction using only a BHQ™-Pulsar™ 650 probe, the instrument will not detect the capillary. To compensate for this limitation, our FAM calibration standard may be spiked into any capillary lacking a FAM-labeled probe, including the BHQ-Pulsar 650 singleplex reactions. We recommend the calibration standard be used at a final concentration of 10 nM. A FAM calibration standard should have been included with your first order of BHQ-Pulsar 650 probe. Please contact us if we can provide you with an additional FAM calibration standard.
The LightCycler® 1.2, does not 'seek' detection of FAM fluorescence during the optical optimisation procedure. To resolve detection of the BHQ-Pulsar 650 probe in this model, raise the 'seek' temperature incrementally to values ranging between 50 °C and 95 °C, until an adequate signal is detected.