Gel Visualiation

Author:

Vinay K L

Copyright:

None

Date:

06/10/2022

Purpose

To visualize either DNA extracts, PCR amplicons.

Before casting gel, prepare 10X(Stock)/1X(Working solution) TBE buffer and GelRed solution.

Steps to prepare Stock 10X TBE Buffer (500ml)

  1. Dissolve 54g of Tris and 27.5g of Boric Acid in 400ml in double distilled water (Use magnetic stirrer, it takes overnight)

  2. Add 20ul of 0.5M Disodium EDTA (Na2EDTA) pH 8.0.

  3. Adjust the volume to 500 ml (~20ml).

  4. Store at room temperature.

Steps to prepare GelRed solution

Unlike the traditional way of adding Gelred directly to agarose gel during casting, we pre-mix the loading dye and Gelred.

  1. Prepare the Orange G loading dye by mixing 200mg of dye in 15ml double distilled water and add 5ml of 30% Glycerol.

  2. Add 40ul of GelRed and and 960 ul of OrangeG dye mixture in amber 1.5ml MCT and make sure to wrap the MCT in aluminum foil (GelRed is light sensitive)

NOTE : We do not use EtBr in the lab. If required for one or two gels, do not prepare the gels in the lab. Ask help from the labs that use EtBr and do the entire process there with their consent and at their convenience. We have a bottle for melting EtBr gels at Raju Mukherjee’s lab.

Steps for casting gels

  1. Prepare 1.5% agarose solution in an conical flask, by mixing the following:

    Small gel

    Big gel

    0.75 g agarose powder

    1.5 g agarose powder

    50 mL 1X TBE buffer

    100 mL 1X TBE buffer

  2. Heat up solution in a microwave until solution is homogenous (~ 1 min for small gel, ~ 2.5 min for big gel). Mix by swirling. This should take several (2-3) cycles of microwaving & swirling.

  3. Cool gel to ~ 60°C using a waterbath, running cold water over the flask, or by sitting on the counter and letting it cool until you can touch the glass without it being extremely warm.

  4. While the gel is cooling, set up the gel bed for casting. Make sure the gel bed is level and that the comb is seated correctly.

  5. Once gel in flask has cooled to a reasonable temperature, pour warm gel on to gel bed and wait until gel solidifies (15–20 minutes).

  6. While gel is solidifying, prepare your samples for loading by mixing each DNA samples/PCR prodcuts with 3ul of loading dye and Gelred mixture.

  7. Remove the comb/s from the gel and transfer the gel (on the gel bed) to the gel rig. Orient the gel so that DNA will run through the gel in the correct direction (away from negative [black] terminal and toward positive [red] terminal). If necessary, add 1X TBE buffer to the gel rig so that the gel is completely immersed in buffer.

  8. Load DNA samples and ladder into the wells of the gel.

  9. Place the lid of the gel rig securely. Connect the terminals to the power box. Run the gel at 90–110 V for ~30-45 minutes.

  10. Take a photo of the gel using the gel doc of biology/chemistry common equipment lab.