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Overloaded Sample (> 8000 RFUs)
- Overloaded peaks can lead to poor data as the ABI software cannot accurately track and size the peaks. Also leads to spectral overlap.
- SOLUTION: Increase dilution factor for problematic dye. Ideal signal is between 300-3000 RFUs.
- Here, the BLUE dye is overloaded (>8000 RFUs) to the point that it is "pulling up" signal in the GREEN (2000 RFUs) and YELLOW (1000 RFUs) ranges. You must be careful to recognize these other peaks as false artifacts of the high BLUE signal.
- SOLUTION: Increase dilution factor for the problematic dye. Ideal signal is between 300-3000 RFUs.
- Definition: Multiplex PCR is when multiple loci are amplified in the same PCR reaction: one template is in the presence of multiple primer pairs.
- Multiplex PCR can save money as the cost for components of the master mix (dNTPs, Taq polymerase, etc.) are minimized to a single reaction.
- Multiplex PCR can be problematic if different loci amplify with different robustness. In a multiplex reaction it is possible for one locus to become overloaded before another locus achieves enough concentration to be identified in capillary electrophoresis of the sample.
- Prior to attempting multiplex PCR it is recommended that you perform the PCR reactions for each primer pair independently for a given locus (1-4 samples).
- Submit these samples in a dilution series and determine whether a common dilution will work for these loci.
- If a common dilution can be determined, perform a multiplex PCR with these primer pairs, and submit a set of samples in another dilution series to verify expected results.
- It may be that some primer pairs can be multiplexed and diluted together while others need to be amplified independently and diluted independently before combining to submit to genotyping.
- It is recommended that you perform this before submitting large sets of samples to minimize the cost per data point. If some loci are too strong the data will be problematic due to spectral overlap. If some loci are too weak the data may be absent.
- Example: failing standard
- Example: clogged capillary
Poor Capillary Performance
- This can be caused by non-labeled contaminanting ions in the sample. Sample clean-up prior to submission should be considered.
- If problem is systemic (same capillary over multiple runs), it indicates that the capillary array is getting old and needs to be changed.
No or Very Low Signal
- PCR failure (bad DNA or Taq)
- Too low a dilution
- SOLUTION: Decrease dilution factor. Try New PCR conditions.