Team:USTC CHINA/Notebook/Protocols/Double Digestion

From 2013.igem.org

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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.</br>
<p>1.Performed with Thermo Scientific FastDigest. There are four restriction endonucleases we used in our experiments: EcoRI, XbaI, SpeI, PstI.</br>
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<span>Single Digestion</span></br>
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<span>Double Digestion</span></br>
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2. Mix gently and spin down.</br>
2. Mix gently and spin down.</br>
3. Incubate at 37°C in a heat block or water thermostat for 30 min.</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>
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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Revision as of 16:35, 25 September 2013

Double Digestion

1.Performed with Thermo Scientific FastDigest. There are four restriction endonucleases we used in our experiments: EcoRI, XbaI, SpeI, PstI.
Double Digestion

Volume (μl)
Plasmid DNA Up to 1μg
10×FastDigest Buffe 2
Enzyme1 1
Enzyme2 1
ddH2O To 20
Total 20
2. Mix gently and spin down.
3. Incubate at 37°C in a heat block or water thermostat for 30 min.
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)

notebook

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