Nucleic acid electrophoresis utilizes the same concepts as protein electrophoresis. The denaturant and visualization techniques differ. All nucleic acids are negatively charged. It is not neccessary or desirable to use a charged denaturant such as SDS. Urea is used to denature the DNA or RNA within the gel. Visualization is usually achieved via staining with ethidium bromide. Ethidium bromide intercalates between base pairs and fluoresces. The fluoresence of free (non-bound) ethidium bromide is quenched. Note that ethidium bromide is a carcinogen and that UV light can damage skin and eyes. Gloves and protective eyeware should be used. The UV box has special anti-UV plastic and a safety interlock. Do not try to defeat the interlock.
Glass plates (10 x 20 cm), spacers,
comb, and clamps
Nucleic Acid samples
6.66 mL 30% Acrylamide
3.34 mL 5X TBE
8.41 g Urea
The TA will have prepared the solution above before lab starts. The solution must be heated to dissolve the urea. However warm gel solutions polymerize quickly with unpredictable results, so the solution must be allowed to cool before use.
140 microliters 10% Ammonium Persulfate
7 microliters TEMED
Loading buffer- 2X Dye- 0.25% Bromophenol Blue, 0.25% Xylene Cyanol
1X TBE for Running Buffer
Very similar to SDS PAGE gel. You should be able to assemble gel by yourself. Have the TA inspect gel prior to use.
Add 8 microliters of the Loading Buffer to 10 microliters of your nucleic acid sample
Load onto gel, and separate your nucleic acid fragments by electrophoresis at around 30 Amps, until the dye front approaches the bottom of gel. Do NOT let dye front run off gel.
Stain with Ethidium Bromide for 5 minutes. Visualize on the UV box. Caution: Wear safety glasses around UV light.
Photograph the gel. Do not dispose of the gel until you have ensured that you have a reasonable photograph.