Question

Briefly describe each step in the process of electrophoresis, beginning with placing molecular ladder, PCR product, and control being placed in three wells. End with taking an image of the gel under UV light. Include what is happening in the gel on a molecular level. site best source

Answer 1

Electrophoresis involves running samples under the influence of electricity.
Steps:
1. Prepare the agarose gel (generally 1% agarose gel is used for the separation of DNA fragments; 1% agarose = 1g of agarose in 100 mL of 1X TAE; Boil the solution and allow it to cool; When it is warm add EtBr and carefully place the required comb)
2. Before the gel gets solidified, add 2-3 uL of EtBr. (We can perform post-staining as well)
3. Place the gel in the electrophoresis tank containing the running buffer (1X TAE)
4. Remove the comb carefully. Make sure that wells have formed properly.
5. Take the PCR sample. Add the required amount of DNA loading dye.
6. Load the samples into the gel lanes carefully. Do not overload the sample and make sure that the gel is submerged in the buffer. Load the DNA ladder and controls along with the samples.
7. Connect the gel tank to electricity. Run the gel at 50-100 mAMP for a 30 min (The running time differs with gel percentage and the product size)
8. Observe the gel using tracking dye bands on the gel.
9. When the gel is run for sufficient time, switch off the electricity. Carefully take out the gel.
10. Document the gel using UV-illuminator (Gel-doc). Save the image and analyze the results.

In the gel, DNA fragments are migrated based on their molecular size. Larger fragments migrate slower as compared to smaller fragments.
In the gel, DNA fragments are illuminated as bright bands when exposed to UV light. This due to the intercalating agent EtBr.

References:
1. Agarose gel electrophoresis for the separation of DNA fragments.
Lee PY et al. J Vis Exp. (2012)

2. Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution.
Stellwagen NC et al. Electrophoresis. (2009)

3. Agarose gel electrophoresis.
Koontz L et al. Methods Enzymol. (2013)