Pouring Agarose Gels

Today we used Agarose gels to verify that our DNA isolation was successful. What I learnt about agarose gels:

1. They are very effective in separating large segments of DNA and RNA.
2. They do not have high resolution, i.e., they cannot separate segments which are of very similar sizes.
3. Range of separation: 200 bases to 10 Kilo bases
4. To separate nucleic acids larger than 10 Kb, you need to run them in Pulsed field gels.
5. Always make the agarose in the same buffer as that in which the gel is run.
6. Use of concentrated buffer while making the gel may result in the gel melting during electrophoresis.
7. garose gels are made in a w/v manner - weight of agar in volume of buffer; For example: in order to make 1% Agarose gel, add one gram of agarose to 100ml of buffer.
8. Buffer used by us: 1X TAE (Tris Acetate EDTA)

Protocol: 1% Agarose Gel (Adjust according to percent and volume required)

1. Take a 200 ml beaker.
2. Add 1 gram of agar to 100 ml of 1X TAE, swirl to mix.
3. Microwave, mixing intermittently, till all the agarose is completely dissolved. The solution will appear clear.
4. Cool the solution till the beaker is comfortable to touch. If the agarose is too warm, it might warp the plates.
5. Prepare the plates before heating the agarose solution. Place the comb. Make sure the plates are on a level surface.
6. Pour the agarose solution into the plates.
7. Let sit at room temperature for 30 minutes. If you need the gel quickly, place in the refrigerator for 5 minutes. The gel is then ready to use.
8. Remove the comb prior to use.