I am encountering several issues in my real-time PCR experiments and seeking guidance on troubleshooting:
1. Inconsistent Amplification Curve Overlap: The amplification curves in my real-time PCR experiments do not consistently overlap between samples. What is the problem and how can I address this issue?
2. Discrepancy Between Gradient PCR and real-time PCR amplicon: I performed gradient PCR (with conventional PCR) for my primers and confirmed the amplicon size using a ladder, but when I ran the real-time PCR amplicon on a 2% agarose gel electrophoresis, it did not match my expected amplicon. What could be the problem, and how can I overcome this discrepancy?
3. Melting Temperature Discrepancies: There is a difference between the predicted melting temperature of my amplicon (from software tools like Oligo 7) and the values obtained in real-time PCR melting curve analysis. What might be causing this disparity?
4. Multiple Peaks in Melting Curve Analysis: Despite setting up the annealing temperature for my primer with pooled cDNA, when I use the accepted annealing temperature for all samples, the melting curve shows multiple peaks without any overlap between samples. What could be the issue, and how can I troubleshoot this problem?
I appreciate any insights or suggestions to address these challenges. Thank you for your expertise.
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