UBL Protocol

for

Running Gels (Large Gel Box)

Revised May 2002

Part I: Casting the Gel

1.Ý There are two casting trays that can be used with the large gel box, a 15cm tray and a 25cm tray.Ý The 15cm tray can accommodate 50-60 samples, while the 25cm tray can accommodate about 100 samples.Ý Decide which one you need before proceeding.

ý Gels using the 15cm tray can be cast in the electrophoresis tub.Ý Assemble the components as shown below (be sure that the casting tray is level; adjust with leveling feet if necessary):

....

ý Gels using the 25cm tray must be cast the white gel caster.Ý Assemble the components as shown below (be sure that the casting tray is level; adjust with leveling feet if necessary):

2.Ý Prepare 0.5% ñ 2% agarose w/v gel solution in 1X Tris-Acetate-EDTA (TAE) or 1X Tris-Borate-EDTA (TBE) buffer:

- 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 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 ÝÝÝÝÝÝÝÝÝÝÝ 2.00ÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝÝ 0.01 ñ 0.5

- Determine the amount of agarose (grams) needed to make the desired gel concentration (%) and volume.Ý The 15cm tray requires 185mL of buffer, while the 25cm tray requires 310mL. ÝThe Agarose % Calculator may be of assistance. 

ÝÝÝÝÝÝÝÝÝÝÝ ý For example: a 1.5% gel on the 15cm tray would require 2.8g agarose (1.5% of 185mL is 2.8g).

- Weigh out the agarose, transfer to a 1000mL flask, then add buffer to volume.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 to 70 degrees C (use a thermometer).

4. ÝAdd ethidium bromide to the gel solution:Ý for the 15cm tray, add __uL of 0.476 mg/mL stock;Ý for the 25cm tray, add __uL of 0.476 mg/mL stock.

5.Ý Swirl gently to mix, then pour gel solution into the casting tray.Ý Rinse the flask immediately with hot water followed by DI water.

6.Ý Allow the gel to solidify (usually takes about 15-30 minutes).

7.Ý If you are using the 15cm tray, carefully remove the two black dams, then fill the gel tub with buffer.Ý If you are using the 25cm tray, transfer the gel with the tray to the tub, then fill with buffer. ÝThe buffer should just cover the top of the gel.

8.Ý Carefully remove the well comb(s) by pulling straight up.

Part II: Loading Samples and Running Gel

9.Ý Load samples. Use 4 ñ 6uL of sample with 1 ñ 2uL of loading dye (Do not add loading dye to the BIO 1xx Alu samplesóthey already contain loading dye: simply load 8-10uL.)

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.

10.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 "+")

11.Ý Turn on the Power Pac and set the running voltage with the 'Increase/Decrease' buttons: for TAE buffer, use 140V;Ý for TBE buffer, use 170V.Ý (Be sure the Power Pac is in Voltage Mode-- the light next to the 'V' should be on.)

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

13.Ý Stop electrophoresis ('Star/Stop' button) when the loading dye is about æ of the way down the gel (usually takes 30-60 minutes).

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

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

SUMMARY

	15 cm Tray	25 cm Tray
1X TAE or TBE Buffer	185 mL	310 mL
Agarose (grams)	Varies	Varies
Ethidium Bromide	__uL of 0.476 mg/mL stock	__uL of 0.476 mg/mL stock
Electrophoresis Volrage	140V (TAE), 170V (TBE)	140V (TAE), 170V (TBE)