ABI Prism® 7900HT Sequence Detection System ... - OpenWetWare

Contaminated
Sample Block
Improper or
Damaged Plastics
Any material contaminating the sample block can affect the results. For example,
mineral oil reduces thermal transfer. Residue from writing on reaction plates darkens
the wells, absorbing light.
The sample blocks should be periodically inspected for cleanliness. Sample block
contamination can be visualized by running a background plate and inspecting the
resulting background signal for aberrant peaks above 2500 FSU (see page 7-13). See
page 7-11 for instructions on decontaminating the sample block.
Only ABI PRISM optical grade reaction plates, optical adhesive covers, and
ABI PRISM® optical flat caps should be used with the ABI PRISM 7900HT Sequence
Detection System. The plastics that comprise the optical parts undergo special testing
for the absence of fluorescent impurities. Optical reaction 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 D, “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), which acts as a normalization factor for fluorescent emissions detected in
the samples (see page A-6).
IMPORTANT Non-Applied Biosystems PCR buffers may not contain the ROX passive
reference. 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 4-12.
Troubleshooting 8-7