AGAROSE GEL ELECTROPHORESIS |
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Last Update: December 2006 |
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| PREPARE SOLUTIONS | |
| 1. 10X DNA loading buffer (50 mL): | 25% Ficoll (Type-400) in dH2O (note: 25% Ficoll is dense and will not go into solution readily, just stir for about 1-2 hours). The buffer is ready for use. But it is a very good idea to add a
dye to not only be able to see your sample as you load it on a gel,
but to track what size fragments during the run. Any combination of
these dyes can be used: 0.25% Bromo-Phenol-blue (300 bp); 0.25% Xylene Cyanol (1-2 kb); 0.25% Orage-G (50 bp). Also, EtBr can be added to the buffer or the gel at a concentration of
~1 mL/10 mL of EtBr. I find it to be better to add it directly to the gel to prevent degradation during storage of the buffer |
| 5X TBE (Tris borate) (1 L): | Mix 54 g of Tris base, 27.5 g of boric acid, 20 mL of 0.5M EDTA, pH 8.0, and dH2O to 1 L |
| 1X TBE (running buffer): | Mix 100 mL of 5X TBE with 900 mL of dH2O |
DNA |
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| PROCEDURE | |
| The concentration of agarose needed to resolve the following fragment sizes: | |
| 1.2% = 100 bp - 5 kb | |
| 2% = 100 bp - 2 kb | |
4% = 20 bp - 500 bp |
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| 1. Mix the desired amoune of agarose with 1X TBE in a flask. For a 1% gel, add 1 g of agarose to 100 mL of 1X TBE | |
| 2. Microwave into solution (while microwaving, take flask out of microwave swirl a few times). It is important the the agarose is completely into solution | |
| 3. Add EtBr if desired | |
| RECOMMENDED: add EtBr to the gel, this way it is quicker to visualize the DNA fragments right after the gel run | |
4. Pour gel into agarose gel set up |
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| 5. Wait until the gel solidified (~1 hr) | |
| 6. Mix DNA samples with loading buffer (1 mL of 10X loading buffer for each 9 mL of DNA sample) | |
| 7.Carefully load DNA samples into the wells of the agarose gel | |
| 8. Using 1X TBE as running buffer, run the agarose gel (100 V is typically more than enough) | |
| 9. Visualize the DNA bands on a UV box or Immaging system | |
RNA |
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| For RNA gel electrophoresis, see protocol Northern blot (113) | |
| The typical bands obtained from total RNA samples are: | |
| 3.3 kb (28S) | |
| 2.9 kb (23S) | |
| 1.8 kb (18S) | |
| 1.3 kb (16S) | |
| 0.2 kb (5S) | |
| The presence of distinct, sharp bands is indicative of good RNA (no degradation) | |