THE BIOTECHNOLOGY
EDUCATION COMPANY
EDUCATION COMPANY
Troubleshooting Guide:
PCR & Electrophoresis
| PROBLEM: | CAUSE: | ANSWER: |
|---|---|---|
| There is very little liquid left in tube after PCR. | Sample has evaporated. | Make sure the heated lid reaches the appropriate temperature. |
| If your thermal cycler does not have a heated lid, overlay the PCR reaction with wax. | ||
| Make sure students close the lid of the PCR tube properly. | ||
| Pipetting Error | Make sure students pipet 20 µL primer mix and 5 µl Lambda DNA template into the PCR tube. | |
| There's not enough sample in my QuickStrip | The QuikStrip has dried out. | Add 40 µL water, gently pipet up and down to mix before loading. |
| The ladder, control DNA, and student PCR products are not visible on the gel | The gel was not prepared properly. | Ensure that the electrophoresis buffer was correctly diluted. |
| Gels of higher concentration (>0.8%) require special attention when melting the agarose. Make sure that the solution is completely clear of "clumps" and glassy granules before pouring gels. | ||
| The gel was not stained properly. | Repeat staining. | |
| Malfunctioning electrophoresis unit or power source. | Contact the manufacturer of the electrophoresis unit or power source. | |
| After staining the gel, the DNA bands are faint. | The gel was not stained for a sufficient period of time. | Repeat staining protocol. |
| After staining, the ladder is visible but no PCR products are present. | PCR amplification was unsuccessful. | Repeat PCR with fresh PCR EdvoBeads™ and primers. |
| Ensure that the thermal cycler has been properly programmed. | ||
| After staining, the ladder and control PCR products are visible on gel, but some student samples are not present. | Wrong volumes of DNA and primer added to PCR reaction. | Practice using pipettes. |
| After staining, the ladder and control PCR products are visible on gel, but some student samples are not present. | Wrong volumes of DNA and primer added to PCR reaction. | Practice using pipettes. |
| DNA bands were not resolved. | Tracking dye should migrate at least 3.5 cm (if using a 7x7 cm tray), and at least 6 cm (if using a 7x14 cm tray) from the wells to ensure adequate separation. | Be sure to run the gel at least 6 cm before staining and visualizing the DNA (approximately one hour at 125 V). |
| DNA bands fade when gels are kept at 4˚C. | DNA stained with FlashBlue™ may fade with time. | Re-stain the gel with FlashBlue™. |