Topmusicanswers
Troubleshooting PCR and RT-PCR Amplification
| Problem | Possible cause |
|---|---|
| No bands on gel | Extension time was too short |
| Thermal cycler was not at correct temperature | |
| Extra, nonspecific bands on gel | Primers hybridized to a secondary site on the template |
| DNA contamination was introduced in primers or buffers |
- What causes PCR failure?
- How do you troubleshoot a PCR problem?
- What would you do if the PCR amplification of a sample has failed?
- What happens when you add too much primer to PCR?
What causes PCR failure?
Usually the first thing researchers do is blame a faulty enzyme or reagent when an experiment fails but with PCR this is actually less likely to be the cause for failure. More often deeper internal problems such as primer design, thermocycler parameters, or nonspecific binding to other template sequences are the cause.
How do you troubleshoot a PCR problem?
Check amplification length capability of the selected DNA polymerases. Use DNA polymerases specially designed for long PCR. Choose DNA polymerases with high processivity, which can amplify long targets in a shorter time. Reduce the annealing and extension temperatures to help primer binding and enzyme thermostability.
What would you do if the PCR amplification of a sample has failed?
If an amplification reaction fails and you suspect the DNA template is contaminated with an inhibitor, add the suspect DNA preparation to a control reaction with a DNA template and primer pair that has amplified well in the past .
What happens when you add too much primer to PCR?
The use of higher concentrations of primers can have the following effects: If the primers are capable of forming dimers, raising their concentration only results in the creation of primer-dimers and does not improve the amplification of the desired PCR product.
