For the following questions 3 - 7, consider what specific effect the following mistakes would have on the results if everything else was done correctly and electrophoresis was completed. Simply stating that the results would be "incorrect" or "messed up" is not acceptable to earn full credit.

Question

Question 3 (2 points)

The loading buffer that was provided did not have SafeGreen. SafeGreen was not used in any other step of the procedure.

Question 3 options:
Question 4 (2 points)

A DNA ladder was not loaded on the gel.

Question 4 options:
Question 5 (2 points)

The loading buffer was improperly prepared, and the glycerol was not added to it.

Question 5 options:
Question 6 (2 points)

The gel was placed in the electrophoresis chamber with the wells closest to the positive electrode.

Question 6 options:
Question 7 (2 points)

The gel was supposed to be run for 30 minutes, but someone forgot about it and accidentally ran it for 3 hours.

Answer 1

Question 3:

Effect: Without SafeGreen in the loading buffer, the DNA bands in the gel would not be visible under UV light after electrophoresis. This means that while the electrophoresis itself may still occur, no visualization of the DNA can be performed, making it impossible to interpret the results or assess the presence, absence, or size of the DNA fragments.

Question 4:

Effect: If a DNA ladder (or marker) was not loaded onto the gel, it would be impossible to determine the size of the DNA fragments present in the samples. The DNA ladder serves as a reference for estimating the sizes of the unknown DNA bands. Without it, one would lack a baseline for comparison, leading to difficulties in analyzing fragment sizes and determining how many PCR products were generated or if any significant banding patterns exist.

Question 5:

Effect: If the loading buffer was prepared without glycerol, the samples may not sink into the wells properly during loading, as glycerol helps to give density to the mixture. This would likely result in the samples diffusing into the buffer and not staying in the confines of the wells, leading to poor resolution and smeared bands. The intended separation of DNA fragments would not occur correctly, producing results that could be misleading or uninterpretable.

Question 6:

Effect: Placing the gel in the electrophoresis chamber with the wells closest to the positive electrode would cause the DNA samples to migrate in the wrong direction. DNA is negatively charged due to its phosphate backbone and is expected to move toward the positive electrode. If the gel is flipped, the DNA will move out of the wells and potentially out of the gel altogether, leading to a complete loss of the sample and resulting in no observable bands.

Question 7:

Effect: Running the gel for an extended time of 3 hours instead of the intended 30 minutes can lead to several problems, such as excessive diffusion of the DNA bands. Overrunning may cause the bands to become too broad and diffuse, making it difficult to accurately interpret results. Additionally, the DNA might migrate out of the gel, leading to a complete loss of the visible bands. This prolonged run time may create a smeared appearance, obscuring distinct bands and hindering the ability to assess the size and integrity of the DNA samples.