Real-Time PCR Troubleshooting

PCR users have sometimes trouble with their real-time PCR experiments. Their real-time PCR curves give no signal or they have complication with signal in negative control. We will try to help users with the main reasons of your PCR troubles and how to solve them.

Do you have no signal?

If there is no signal in your real-time PCR results, there could be some of the following issues:

There is no template in PCR. It looks like a trivial cause of no signal in your PCR, but always check again that you have added the template to your PCR. This can sometimes help you to save time and nerves in solving the problem.
There are inhibitors in PCR. It is possible that your template contains inhibitors that cause poor PCR, for example salts, SDS, phenol, plant and human tissues or fluids. The solution: It is appropriate to use additives (BSA, DMSO, Betaine, etc.) to enhance your PCR. Next option is usage of internal positive control that can check the correctness of PCR.

Recommended products:
gb IPC PCR Master Mix
gb OneStep IPC RT-qPCR kit

There is problem with Probe quality or design. The probe could be partially degraded because of high amount of freeze-thaw cycles or wrong long-term storage. The probes synthetized in Generi Biotech may have up to 50 freeze-thaw cycles. It is also possible that the probe is not designed properly and doesn’t bind to the amplified product.
Oligos and probes
There is an issue with thermal profile optimization. Be sure to have adequate time and temperature for all steps of your PCR.

Do you have signal in negative control?

If there is positive signal in negative control (or no template control), there could be some of the following issues:

There is a possibility that you have added the template into your NTC reaction. Please repeat the analysis to exclude this problem.
There is a problem with primers and probes design. It is possible that your primers and probes generate dimers and your PCR gives you false positive signal in your analysis. Try to check your PCR product by electrophoresis or by melting analysis.

gb Easy PCR Master Mix

gb Easy PCR Master Mix consists of hot-start Taq DNA polymerase, reaction buffer, dNTP and MgCl2. The Taq polymerase is chemically modified DNA polymerase from Thermus aquaticus. This polymerase is completely inactive at room temperature but it is rapidly activated during the initial denaturation step of PCR. The product falls into a group of TaqMan Real-Time PCR Master Mixes to provide probe-based qPCR.

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gb HRM PCR Master Mix

gb HRM PCR Master Mix falls into a group of Dye-Based Real-time PCR Master Mixes. The product is intended for amplification of DNA with subsequent High Resolution Melting (HRM) analysis. HRM analysis is an efficient melt curve analysis method for genotyping, mutation scanning prior to sequencing analysis, DNA mapping, species identification or DNA methylation analysis etc.

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