TBE Urea Page Gel

Urea PAGE separations are run in denaturing conditions. This is useful for analysis of RNA or ssDNA, which may have significant secondary structure in non-denaturing conditions.

Contents

1. Recipe
2. Procedure
3. Running and imaging

1. Recipe

Recipe for 6-8 gels.

Resolving Layer	5%	6%	8 %	10%	12%	15%
5X TBE (ml)	5	5	5	5	5	5
40% Acryl (ml)	6.25	7.5	10.0	12.5	15.0	18.75
10% APS (ml)	0.5	0.5	0.5	0.5	0.5	0.5
TEMED (ul)	20	20	20	20	20	20
Urea (g)	25	25	25	25	25	25
H2O (ml)	to 50	to 50	to 50	to 50	to 50	to 50

*** The Urea makes it hard to give a definitive amount of water to add.

2. Procedure

1. Prepare the glass plates, combs and casting station before you combine any chemicals. Read the manual to learn how to use the casting system.
2. Adding a 1000 mL pipette tip between the casting station clip will help keep the gel from leaking (it can still happen though).
3. Weigh the Urea (25g) in a 50 mL conical tube.
4. Combine 5x TBE and Acrylamide in the 50 mL tube. Add water to near 47.5 mL on the conical. Mix throughly and then incubate at 37C to solubilize the Urea (it takes a while).
5. Once the Urea is dissolved, allow the solution to return to room temperature. If you do not allow the solution to cool, the polymerization occurs too quickly to pipet into the glass.
6. Add APS and TEMED. Fill the conical to 50 mL with water. Invert to mix the solution.
7. Fill the glass plate to the brim (no stacking layer for Urea-PAGE gels). Insert the comb and let the gel polymerize.
8. Wait ~20 minutes for the gel to solidify.
9. Wrap the gel plates with a damp paper towel and place the gel stack into a plastic bag.
10. Label the bag with gel%/Urea-PAGE date made, and initials.

3. Running and imaging

1. Run TBE gel in 0.5X TBE buffer http://cshprotocols.cshlp.org/content/2006/1/pdb.rec8458
2. Wash the gel lanes with 0.5X TBE using a needle attached to a 30 mL syringe (you will see Urea being washed out).
3. Pre-run the gel for 30 minutes at 120V. This allows the gel to equilibrate temperature.
4. Mix samples 1:1 with 2X Formamide Buffer. Boil RNA at 95C for 5 minutes. Place immediately on ice.
5. Again wash the gel lanes with 0.5X TBE before loading. This will remove Urea before running the gel (you will again see Urea being washed out).
6. Run the gel at 120V for 50-55 minutes. The dye runs at tens of RNA nucleotides, therefore, you need to run the gel further than the dye indicates.
7. Remove the gel from the casing and soak for 15-20 minutes in 1X SYBR Gold in 0.5X TBE.
8. Pour off SYBR Gold. Wash with 0.5X TBE for 5-10 minutes.
9. Image the gel in UV Box.