Cong T. Trinh:electrophoresis

DNA Gel Electrophoresis

Gel Preparation

• 60mL of 1x TAE Buffer

The amount of agarose to use in your gel depends on the DNA in question. Use the following table as a rough guide:

Agarose Concentration (g/100mL)	Optimal DNA Resolution (kb)
0.5	1 - 30
0.7	0.8 - 12
1.0	0.5 - 10
1.2	0.4 - 7
1.5	0.2 - 3

• .54g is typically used for the small gels, 1.08g for the large gels

-Microwave the Agarose and TAE buffer mixture for ~1:15 min. -Let cool for ~5 min so that you can touch the beaker with your bare hand. -Add 3.5-6 μL of Ethidium Bromide -Pour the agarose solution into the sealed gelbox. Carefuly pop or shove to the side any bubbles with the gel comb, place the gel comb to one end of the gel, and let it cool for about 30 minutes, until the gel is solid.

Buffers

• TAE - better resolution of fragments >4kb;
• TBE - better resolution of 0.1-3kb fragments; TBE is better suited for high-voltage (>150V) electrophoresis than TAE because of its higher buffering capacity and lower conductivity;
• SB - Sodium borate - low heat generation with good resolution. Allows for higher voltage (250-200V) electrophoresis without melting.

Notes

• If you are getting unexpected bands on your gel you may want to look at the common issues in agarose gel electrophoresis page.
• If you have no experience with gel electrophoresis or are explaining it to someone new, here is a cute java demo of what happens.

Gel Loading

-20μL of Ladder --Be sure to use the appropriate size ladder for the expected product band length -20μL of PCR product --Be sure the a loading dye has been added to the DNA -Run @125v for ~1hr (varies depending on expected product size) -