Silver:Restriction digest protocol: Difference between revisions

Revision as of 00:41, 23 September 2009

<html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="restriction enzyme1">restriction enzyme1 <tab><div style="margin-right: 600px;">(20 units/µl)</div></a></li><li> <a name="restriction enzyme2">restriction enzyme2 <tab><div style="margin-right: 600px;">(20 units/µl)</div></a></li><li>10X restriction buffer</li><li>10X BSA</li><li>PCR product DNA</li><li>distilled H2O</li><li> <a name="BioBrick vector">BioBrick vector <tab><div style="margin-right: 600px;">(BBa_V0002 or BBa_V0100)</div></a></li><li> <a name="CIP">CIP <tab><div style="margin-right: 600px;">(10 units/µl)</div></a></li></ul><h2>Parameters:</h2><ul type="circle"><li>V - Volume of PCR product DNA obtained (µl).</li><li>C - Concentration of BioBrick vector (µg/µl).</li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Reaction tubes</li></ul><h2>Steps:</h2><ol><li>Digestion of PCR product Use the following table as a checklist for preparing the reaction: <table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td> </td><td>PCR product DNA</td><td>10X BSA</td><td>10X restriction buffer</td><td>restriction enzyme1</td><td>restriction enzyme2</td><td>distilled H2O</td></tr></thead><tbody><tr><td>Restriction Digest - PCR product</td><td>V</td><td>3.5 µl</td><td>3.5 µl</td><td>0.2 µl</td><td>0.2 µl</td><td>Make up the volume to 35 µl</td></tr></body></table>Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume. </li><li>Incubate at 37°C for at least 1 hr. Incubation overnight is better. </li><li>Purify digested insert using PCR product purification kit. </li><li>Digestion of BioBrick vector: Recommended procedure Use the following table as a checklist for preparing the reaction: <table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td> </td><td>BioBrick vector</td><td>10X BSA</td><td>10X restriction buffer</td><td>restriction enzyme1</td><td>restriction enzyme2</td><td>distilled H2O</td></tr></thead><tbody><tr><td>Restriction Digest - vector</td><td>7/C</td><td>1 µl</td><td>1 µl</td><td>0.2 µl</td><td>0.2 µl</td><td>Make up the volume to 10 µl</td></tr></body></table>Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume. </li><li>Incubate at 37°C for 12 hrs(overnight). </li><li>Add 0.1 µl of <a href="#CIP" >CIP</a>. Incubate at 37°C for 1 hr. </li></ol></html>

Silver:Restriction digest protocol: Difference between revisions

Revision as of 00:41, 23 September 2009

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