Question

In a manual sequencing reaction, the sequencing ladder on the polyacrylamide gel is very bright and readable at the bottom of the gel, but the larger (slower migrating) fragments higher up are very faint. What is the most likely cause of this observation? How might it be corrected?

Answer 1

The most likely cause of the observation is the slower migration of larger fragments. This can be corrected by optimizing gel concentration, voltage application, and gel temperature.

Step-by-step explanation:

The most likely cause of the observation where the sequencing ladder on the polyacrylamide gel is very bright and readable at the bottom but faint higher up is that the larger fragments are migrating slower and getting stuck or diffusing away as they migrate.

This can be due to various factors such as uneven gel concentration or density, insufficient or uneven voltage applied, or gel temperature being too high.

To correct this, the gel concentration or density can be optimized, ensuring uniform voltage application, and maintaining proper gel temperature. This can improve the migration of larger fragments and make them more visible on the gel.