UBL Protocol

for

Running Gels (Small Gel Box)

Revised May 2002

1.Ý Assemble the gel box assembly according to the pictures below (leave the lid off for now).Ý Note that there are two different size combs available (a __well and a __ well); choose based on the number of samples you have to run.

ý Make sure that the gel tub is level.Ý Check with a leveling bubble and adjust by turning the leveling screws if necessary.

2.Ý Prepare a 20mL gel solution (0.5% ñ 2% agarose w/v) in 1X Tris-Acetate-EDTA (TAE) buffer.

- Determine the amount of agarose (grams) needed to make the desired gel concentration (%) in 20mL.Ý Example: for a 1% gel, use 0.2 gram of agarose (1% of 20mL is 0.2g.). Use the Agarose % Calculator if you are not sure.

- Optimal gel concentration depends on the size of the DNA to be separated.Ý Consult the following table:

Gel Concentration (%)ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝ DNA Size (Kb)

ÝÝÝÝÝÝÝÝÝÝÝ 0.50ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝ 1 - 30

ÝÝÝÝÝÝÝÝÝÝÝ 0.75ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝ 0.8 ñ 12

ÝÝÝÝÝÝÝÝÝÝÝ 1.00ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝ 0.5 ñ 10

ÝÝÝÝÝÝÝÝÝÝÝ 1.25ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝ 0.4 ñ 7

ÝÝÝÝÝÝÝÝÝÝÝ 1.50ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝ 0.2 ñ 3

ÝÝÝÝÝÝÝÝÝÝÝ 2.00ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝ 0.01 ñ 0.5

- Weigh out the agarose, transfer to a flask, then add buffer (20mL 1X TAE).Ý Microwave on HIGH for ~20 seconds, then swirl to mix.Ý Repeat until the gel is completely dissolved (solution should be clear!)

3.Ý Allow the gel solution to cool for 5 minutes, or until the flask is comfortable to the touch.

ý Do not let the gel sit for too long or it will solidify!

4.Ý Pour the gel solution into the center of the gel tub, then rinse the flask immediately with hot water followed by DI water.

5. ÝAllow the gel to solidify for 10-15 minutes.

6.Ý Carefully remove the two black dams, then fill the gel tub with 1X TAE.Ý The buffer should just cover the top of the gel.

7.Ý Carefully remove the well comb by pulling straight up.

ý Do not remove the comb at an angle or you may damage the wells.

8.Ý Load your samples into the wells.Ý

- For the 'small' wells, use 2 ñ 3uL of sample with 1 ñ 2uL of loading dye.

- For the 'large' wells, use 4 ñ 6uL of sample with 1 ñ 2uL of loading dye.

Tips on Loading Gels

1. If you can, use the long, skinny tips (the red ìLî tips). They are easier to insert into the wells.

2. Before loading, be sure that the tip does not have any bubbles in it (clear spots). If there are air bubbles, the air will force your sample out of your well and into the buffer. (If you pipet your sample carefully, there should be no air bubbles!)

3. Donít stick the tip all the way down into the well. You want to have room for the sample to flow. If you are loading a large sample, raise the tip slowly as you inject.

9.Ý Place the lid on the gel tub and connect the cables to the Power Pac (they are color-coded; black is "-" and red is "+")

10.Ý Turn on the Power Pac and set the running voltage (usually 75-90V) with the 'Increase/Decrease' buttons. Be sure the Power Pac is in Voltage Mode-- the light next to the 'V' should be on.

11.Ý Begin electrophoresis by pressing the 'Start/Stop' button.

12.Ý Stop electrophoresis ('Star/Stop' button) when the loading dye is about æ of the way down the gel (usually takes 35-45 minutes).

ý You can also preset the running time by changing to Time Mode (press the 'V/A/Time selection button' until the light next to the clock symbol is on) then setting the time with the 'Increase/Decrease' button.

13.Ý Stain the gel in ethidium bromide for 10 minutes.

14.Ý Rinse the gel in water for 10 minutes.

15.Ý Visualize the gel in the Gel Doc. Protocol

16.Ý Discard or recycle the used TAE buffer, then rinse the gel box components with DI water and put away.