ELECTROPHORESIS OF DNA IN POLYACRYLAMIDE GELS
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ELECTROPHORESIS OF DNA IN POLYACRYLAMIDE GELS
Gel SizesSmall: 165 x 130 mm
Medium: 165 x 200 mm
Large: 165 x 260 mm
5% Analytical Gels
| Reagent | <center><b><font>1 mm Small</font> </b> </center> | <center><b><font>1 mm Medium</font> </b> </center> | <center><b><font>1 mm Large</font> </b> </center> |
| 10X TBE Buffer (ml) | <center><font>3</font> </center> | <center><font>3.5</font> </center> | <center><font>5</font> </center> |
| 40% bis/acrylamide (ml) | <center><font>3.75</font> </center> | <center><font>4.4</font> </center> | <center><font>6.2</font> </center> |
| Water (ml) | <center><font>17.25</font> </center> | <center><font>20.1</font> </center> | <center><font>28.8</font> </center> |
| 50% Glycerol (ml) | <center><font>6</font> </center> | <center><font>7</font> </center> | <center><font>10</font> </center> |
| APS (mg) | <center><font>34</font> </center> | <center><font>40</font> </center> | <center><font>57</font> </center> |
| TEMED (ul) | <center><font>12.8</font> </center> | <center><font>15</font> </center> | <center><font>21.4</font> </center> |
| Total Volume (ml) | <center><font>30</font> </center> | <center><font>35</font> </center> | <center><font>50</font> </center> |
With other concentrations of a bis/acrylamide stock substitute:
| 25% bis/acrylamide (ml) | 6.0 | 7.0 | 10.0 |
| Water (ml) | 15.0 | 18.5 | 25.0 |
| 30% bis/acrylamide (ml) | 5.0 | 5.8 | 8.3 |
| Water (ml) | 16.0 | 18.7 | 26.7 |
| 50% bis/acrylamide (ml) | 3.0 | 3.5 | 5.0 |
| Water (ml) | 18.0 | 21.0 | 30.0 |
5% BAC Cross-linked Polyacrylamide Gels
| Reagent | 1 mm Medium | 1 mm Long | 4 mm Small | 4 mm Medium |
| 10x TBE Buffer (ml) | <center><font>8</font> </center> | <center><font>10</font> </center> | <center><font>16</font> </center> | <center><font>20</font> </center> |
| Water (ml) | <center><font>14</font> </center> | <center><font>17.5</font> </center> | <center><font>28</font> </center> | <center><font>35</font> </center> |
| 50% Glycerol (ml) | <center><font>8</font> </center> | <center><font>10</font> </center> | <center><font>16</font> </center> | <center><font>20</font> </center> |
| 20% bac/acrylamide (ml) | <center><font>10</font> </center> | <center><font>12.5</font> </center> | <center><font>20</font> </center> | <center><font>25</font> </center> |
| APS (mg) | <center><font>16</font> </center> | <center><font>20</font> </center> | <center><font>32</font> </center> | <center><font>40</font> </center> |
| TEMED (ml) | <center><font>200</font> </center> | <center><font>250</font> </center> | <center><font>400</font> </center> | <center><font>500</font> </center> |
| Total Volume (ml) | <center><font>40</font> </center> | <center><font>50</font> </center> | <center><font>80</font> </center> | <center><font>100</font> </center> |
Pre-run gels for 30 minutes at 80V. Rinse the wells with running buffer. Load and electrophorese samples at 80V until dyes separate, then boost to up to 150 V. Medium sized polyacrylamide gels can be run overnight at 55-60 V to see all phi-X/Hae III cut molecular weight standards on the gel.
7.5% Mini (10 x 8.2 cm) Polyacrylamide Gels
SEQUENCING GELS (8% Polyacrylamide, 8 M Urea)
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| Reagent | 40 cm gel | 80 cm gel | 1 m gel |
| Urea (g) | <center><font>48</font> </center> | <center><font>86.5</font> </center> | <center><font>120</font> </center> |
| Water (ml) | <center><font>37</font> </center> | <center><font>66.5</font> </center> | <center><font>92.4</font> </center> |
| 50% bis/acrylamide (ml) | <center><font>16</font> </center> | <center><font>28.8</font> </center> | <center><font>40</font> </center> |
| 10x TBE Buffer (ml) | <center><font>10</font> </center> | <center><font>18</font> </center> | <center><font>25</font> </center> |
| 10% (w/v) APS (ml) | <center><font>0.668</font> </center> | <center><font>1.2</font> </center> | <center><font>1.67</font> </center> |
| TEMED (ml) | <center><font>50</font> </center> | <center><font>85</font> </center> | <center><font>118</font> </center> |
| Total Volume (ml) | <center><font>100</font> </center> | <center><font>180</font> </center> | <center><font>250</font> </center> |
With other concentrations of a bis/acrylamide stock substitute:
| 30% bis/acrylamide(ml) | <center><font> </font> </center> | <center><font>48</font> </center> | <center><font>66.7</font> </center> |
| Water (ml) | <center><font> </font> </center> | <center><font>47.3</font> </center> | <center><font>65.7</font> </center> |
| 40% bis/acrylamide (ml) | <center><font> </font> </center> | <center><font>36</font> </center> | <center><font>50</font> </center> |
| Water (ml) | <center><font> </font> </center> | <center><font>59.3</font> </center> | <center><font>82.4</font> </center> |
<center><b><u><font>RECIPES</font> </u> </b> </center>
- 10X TBE (1M Tris, 1M Boric Acid, 20mM EDTA, pH 8.3)
- 242.2 g Tris
- 123.66 g boric acid
- 14.89 g EDTA
- Adjust pH to 8.3. QS to 2 liters. Autoclave. (Biotechniques
10:182, 1991 claims that filtering up to a 20x TBE solution through 0.2 - 0.45µ cellulose acetate or cellulose nitrate filters prevents formation of precipitants during long-term storage. The solution may be reautocalved to dissolve precipitates that form.)
- 50% Glycerol
- 25 ml 100% glycerol
- 25 ml water
- Autoclave. Concentrated glycerol is quite viscous. Be sure all of it has been transferred from the stock bottle.
- 30% Acrylamide Solution (0.8% bis)
- 60 g acrylamide
- 1.6 g bis
- Heat to dissolve in approximately 100 ml of water. QS to 200 ml with water and filter with Whatman #1 filter paper into a foil wrapped bottle.
- 40% Acrylamide Solution (19:1 acrylamide:bis)
- 80 g acrylamide
- 4.21 g bis
- Heat to dissolve in approximately 100 ml of water. QS to 200 ml with water and filter with Whatman #1 filter paper into a foil wrapped bottle.
- 50% Acrylamide Solution (19:1 acrylamide:bis)
- 237.5 g (97%) acrylamide
- 12.5 g bis
- Dissolve in 250 ml hot water. Filter to remove debris and wrap in foil.
- 20% Bac-Acrylamide Solution WARNING: wear gloves
- 100 ml solution
- 18.98 g acrylamide
- 1.02 g BAC (N',N'-bis-acrylylcystamine)
- Heat approximately 75 ml of water to almost boiling. Add acrylamide and cover with a watch glass. Mix briefly to dissolve and reheat to near boiling. Add BAC. QS to 100 ml with water. Do not autoclave. Filter sterlize with Nalgene 0.45 or 0.2 micron filter and store in foil wrapped bottle.
7.5% Polyacrylamide Gel Solution
| 40% Bis/Acrylamide Stock Solution | 18.75 ml | 37.5 ml |
| 10x TBE buffer | 10 ml | 20 ml |
| 50% glycerol | 20 ml | 40 ml |
| Water | 51.25 ml | 102.5 ml |
| TOTAL VOLUME | 100 ml | 200 ml |
