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Team:USTC CHINA/Notebook/Protocols/Double Digestion
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<h1>Double Digestion</h1> | <h1>Double Digestion</h1> | ||
| - | <p>1.Performed with Thermo Scientific FastDigest. There are four restriction endonucleases we used in our experiments: EcoRI, XbaI, SpeI, PstI. | + | <p>1.Performed with Thermo Scientific FastDigest. There are four restriction endonucleases we used in our experiments: EcoRI, XbaI, SpeI, PstI.</br> |
| + | <span>Double Digestion</span></br> | ||
| + | <table width="580" border="2"> | ||
| + | <tr> | ||
| + | <td></td> | ||
| + | <td>Volume (μl)</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Plasmid DNA</td> | ||
| + | <td>Up to 1μg</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>10×FastDigest Buffe</td> | ||
| + | <td>2</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Enzyme1</td> | ||
| + | <td>1</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Enzyme2</td> | ||
| + | <td>1</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>ddH<sub>2</sub>O</td> | ||
| + | <td>To 20</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Total</td> | ||
| + | <td>20</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | 2. Mix gently and spin down.</br> | ||
| + | 3. Incubate at 37°C in a heat block or water thermostat for 30 min.</br> | ||
| + | 4. Inactivate the enzyme by adding glycerol DNA loading buffer, or by heating for 5 min at 80°C(only for EcoRI) or 20min at 65°C(only for XbaI)</br> | ||
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Latest revision as of 08:56, 27 September 2013
