Applied Biosystems 7900HT Fast Real-Time PCR System and SDS ...

Chapter 8TroubleshootingImproper orDamagedPlasticsLow CopyTemplatesUse ofNon-AppliedBiosystems PCRReagentsOnly Applied Biosystems optical plates, optical adhesive covers, and optical flat capsshould be used with the Applied Biosystems 7900HT Fast Real-Time PCR System.The plastics that comprise the optical parts undergo special testing for the absence offluorescent impurities. Optical plates are frosted to improve the degree and precisionof light reflection. Bent, creased, or damaged plastics may adversely affect thetransmission of fluorescent signal or prevent proper sealing of a well resulting inevaporation, change in sample volume, and altered PCR chemistry. Make sure to usethe correct plastics and visually inspect each reaction plate before use.Note: See Appendix C, “Kits, Reagents, and Consumables,” for a list of compatibleconsumables and reagents.When amplifying samples that contain very low quantities of nucleic acid (generallyless than 100 molecules), expect lowered precision due to the Poisson distributionand biochemical effects related to binding probabilities. Low copy templates are alsomore susceptible to losses due to non-specific adhesion to plastic wells, pipettor tips,etc. The addition of carrier to the sample, such as yeast tRNA or glycogen, can helpprevent these losses, increasing the precision and sensitivity of the assay.The Applied Biosystems buffer contains an internal passive reference molecule(ROX dye), which acts as a normalization factor for fluorescent emissions detectedin the samples.IMPORTANT! Non-Applied Biosystems PCR buffers may not contain the ROXpassive reference dye. If running non-Applied Biosystems chemistry, be sure to setthe passive reference for your experiment as explained on “Setting the PassiveReference” on page 3-18.8-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User GuideDRAFTSeptember 1, 2004 11:39 am, CH_Trouble.fm