Question

10. You electrophorese a DNA sample containing two linear fragments of DNA, one large and one small. Which one do you expect to find closest to the negative electrode? 11. Why do you not increase the voltage during electrophoresis? It would speed up the process considerably. What if you mistakenly used distilled water instead of TAE buffer for your electrophoresis? Would there be any difference in the outcome? 12. 13. What does ethidium bromide do?

Answer 1

10. DNA is negatively charged due to presence of phosphate groups. Hence, it will migrate from negatively charged electrode to positively charged electrode in an electric current, during gel electrophoresis. The migration of DNA fragment depends on molecular weight. Larger fragments will move slowly as they are impeded by the pores in the gel. On the other hand, smaller fragments (low molecular weight) can easily pass through gel pores and move towards the positive charged electrode. Hence, the larger fragment will be seen near negatively charged electrode. The smaller fragment will be seen near the positively charged electrode.

Right choice: Large fragment.

11. Increasing the voltage will speed up the reaction but it will cause heating of the gel. This will cause the gel to melt at high voltage. Running the gel at lower voltage will allow slow migration of the bands and also prevents the heating of gel. Slow migration also allows better separation of the fragments based on molecular weight. High voltage may also break the DNA that is being separated due to heat.

12. The TAE buffer is composed of Tris, acetic acid and EDTA. Its major function is to maintain pH of the medium during gel electrophoresis. It also has ions that help in generating electric current. Distilled water lacks ions and hence cannot generate the electric current. This will prevent movement of DNA through the gel. DNA will remain in the wells if distilled water instead of TAE buffer.

13. Ethidium bromide is a DNA interacting fluorescent dye. It binds to DNA between nitrogenous bases and will fluoresce when exposed to UV light. Ethidium bromide will emit orange fluorescence when exposed to UV light. Thus, it will detect the different DNA fragments that have migrated through the gel. The DNA fragments can then be cut out from the gel based on Ethidium bromide fluorescence, after which DNA elution can be performed.