1. What is the size of the fragment that contains the Alu insertion? Without the

Question

1. What is the size of the fragment that contains the Alu insertion? Without the Alu insertion?

2. Describe the two functions of loading dye.

3. What is the purpose of using a marker or ladder during electrophoresis?

4. Suppose you have 4 fragments of 500bp each. How many bands would appear on the gel?

5. Describe the quick stain protocol that you will be following to visualize the DNA.

6. Suppose you run a gel, and you are concerned that you may have ‘lost’ some of the smallest fragments due to them running off the gel. What two things can change in ensure that these would remain on the gel the next time you repeat the experiment?

Explanation / Answer

Answer:

(Question 1 requires more information about the sequence or size of the DNA fragments)

2. The two functions of loading dye are:

i. Tracking the DNA sample during the gel run:

The loading dye contains bromophenol blue (may also contain xylene cyanol) which helps to track the DNA during the gel run. Bromophenol blue and xylene cyanol can be used singly or in combination to track the extent of DNA migration. If used in combination, the DNA is generally present in between the two tracking dyes.

ii. DNA sample loading:

Loading dyes contain glycerol which binds to the DNA and makes it havier such that the sample sinks to the bottom of the well.

3. Markers or ladders are a set of standards/ fragments of known sizes which can be used to judge the approximate size of the unknown (test) sample bands.

4. A single band will appear on the gel since all the 4 bands will overlap with each other in the gel due to their equivalent sizes.

5. The quick stain protocol for visualizing the DNA has been discussed below:

i. The gel from the gel tray should be removed carefully and put into the staining tray.

ii. Approximately 120 ml of 100x stain should be poured into the tray to completely submerge the gel. More stain should be poured if required.

iii. After staining for about 2-3 minutes, the stain should be poured off which can be stored for later use.

iv. The gel should be transferred to 500–700 ml of clean, warm (40–55°C) tap water and shaken gently for 10 to 15 minutes to rinse.

v. The gel should then be transferred to a larger container containing 500–700 ml of clean water and sheken in a rocker for 15 minutes.

vi. A second washing step should then be performed similar to step v.

vii. The water can now be poured off and examined for the presence of bands.

6. The following two changes can be made to ensure that the fragments remain in the gel:

i. Increasing the percentage of agarose in the gel or the length of the gel (dimensions):

Increasing the percentage of agarose in the gel would allow the small fragments to be reatined in the gel due to its smaller pore size and slower rate of migration, while increasing the length of the gel would increase the duration of migration.

ii. Decreasing the voltage during the run would help to slow the migration of the smaller DNA fragment and thus prevent their run off.

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