DRAFT
September 1, 2004 11:39 am, CH_Trouble.fm
Chapter 8 Troubleshooting
8-8 Applied Biosystems 7900HT Fast Real-Time PCR System and SDS Enterprise Database User Guide
Improper or
Damaged
Plastics
Only Applied Biosystems optical plates, optical adhesive covers, and optical flat caps
should 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 of
fluorescent impurities. Optical plates are frosted to improve the degree and precision
of light reflection. Bent, creased, or damaged plastics may adversely affect the
transmission of fluorescent signal or prevent proper sealing of a well resulting in
evaporation, change in sample volume, and altered PCR chemistry. Make sure to use
the correct plastics and visually inspect each reaction plate before use.
Note: See Appendix C, “Kits, Reagents, and Consumables,” for a list of compatible
consumables and reagents.
Low Copy
Templates
When amplifying samples that contain very low quantities of nucleic acid (generally
less than 100 molecules), expect lowered precision due to the Poisson distribution
and biochemical effects related to binding probabilities. Low copy templates are also
more 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 help
prevent these losses, increasing the precision and sensitivity of the assay.
Use of
Non-Applied
Biosystems PCR
Reagents
The Applied Biosystems buffer contains an internal passive reference molecule
(ROX
™
dye), which acts as a normalization factor for fluorescent emissions detected
in the samples.
IMPORTANT! Non-Applied Biosystems PCR buffers may not contain the ROX
passive reference dye. If running non-Applied Biosystems chemistry, be sure to set
the passive reference for your experiment as explained on “Setting the Passive
Reference” on page 3-18.
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