Silver:Restriction digest protocol

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(New page: <html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="restriction enzyme1">restriction enzyme1 <i><br><tab><div style="margin-right: 600px;">(20 units/µl)</div></i></a></li><l...)
Current revision (03:07, 20 November 2009) (view source)
 
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="restriction enzyme1">restriction enzyme1 <i><br><tab><div style="margin-right: 600px;">(20 units/µl)</div></i></a></li><li> <a name="restriction enzyme2">restriction enzyme2 <i><br><tab><div style="margin-right: 600px;">(20 units/µl)</div></i></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 <i><br><tab><div style="margin-right: 600px;">(BBa_V0002 or BBa_V0100)</div></i></a></li><li> <a name="CIP">CIP <i><br><tab><div style="margin-right: 600px;">(10 units/µl)</div></i></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><p><li><b><font size=3>Digestion of PCR product</font></b><br>Use the following table as a checklist for preparing the reaction:<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>PCR product DNA</font></td><td><font color=#357EC7>10X BSA</font></td><td><font color=#357EC7>10X restriction buffer</font></td><td><font color=#357EC7>restriction enzyme1</font></td><td><font color=#357EC7>restriction enzyme2</font></td><td><font color=#357EC7>distilled H2O</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Restriction Digest - PCR product</font></td><td><b><b><font color=#357EC7>V</font></b></td><td><b><b><font color=#357EC7>3.5 µl</font></b></td><td><b><b><font color=#357EC7>3.5 µl</font></b></td><td><b><b><font color=#357EC7>0.2 µl</font></b></td><td><b><b><font color=#357EC7>0.2 µl</font></b></td><td><b><font color=#357EC7>Make up the volume to <b><font color=#357EC7>35 µl</font></b></font></b></td></tr></body></table><font color = "#800517"><i>Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.</i></font><br></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for at least <b><font color=#357EC7>1 hr</font></b>.<br><font color = "#800517"><i>Incubation overnight is better.</i></font><br></li></p><p><li><font color = "#800517"><i>Purify digested insert using PCR product purification kit.</i></font><br></li></p><p><li><b><font size=3>Digestion of BioBrick vector: Recommended procedure</font></b><br>Use the following table as a checklist for preparing the reaction:<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>BioBrick vector</font></td><td><font color=#357EC7>10X BSA</font></td><td><font color=#357EC7>10X restriction buffer</font></td><td><font color=#357EC7>restriction enzyme1</font></td><td><font color=#357EC7>restriction enzyme2</font></td><td><font color=#357EC7>distilled H2O</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Restriction Digest - vector</font></td><td><b><b><font color=#357EC7>7/C</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></td><td><b><b><font color=#357EC7>0.2 µl</font></b></td><td><b><b><font color=#357EC7>0.2 µl</font></b></td><td><b><font color=#357EC7>Make up the volume to <b><font color=#357EC7>10 µl</font></b></font></b></td></tr></body></table><font color = "#800517"><i>Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.</i></font><br></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>12 hrs</font></b>(overnight).<br></li></p><p><li>Add <b><font color=#357EC7>0.1 µl</font></b> of <a href="#CIP" ><font color=#357EC7>CIP</font></a>.<br>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>1 hr</font></b>.<br></li></p></ol></html>
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="restriction enzyme1">restriction enzyme1 <i><br><tab><div style="margin-right: 600px;">(20 units/µl)</div></i></a></li><li> <a name="restriction enzyme2">restriction enzyme2 <i><br><tab><div style="margin-right: 600px;">(20 units/µl)</div></i></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 <i><br><tab><div style="margin-right: 600px;">(BBa_V0002 or BBa_V0100)</div></i></a></li><li> <a name="CIP">CIP <i><br><tab><div style="margin-right: 600px;">(10 units/µl)</div></i></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><p><li><b><font size=3>Digestion of PCR product</font></b><br>Use the following table as a checklist for preparing the reaction in reaction tube (1):<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>PCR product DNA</font></td><td><font color=#357EC7>10X BSA</font></td><td><font color=#357EC7>10X restriction buffer</font></td><td><font color=#357EC7>restriction enzyme1</font></td><td><font color=#357EC7>restriction enzyme2</font></td><td><font color=#357EC7>distilled H2O</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Restriction Digest - PCR product</font></td><td><b><b><font color=#357EC7>V</font></b></td><td><b><b><font color=#357EC7>3.5 µl</font></b></td><td><b><b><font color=#357EC7>3.5 µl</font></b></td><td><b><b><font color=#357EC7>0.2 µl</font></b></td><td><b><b><font color=#357EC7>0.2 µl</font></b></td><td><b><font color=#357EC7>Make up the volume to <b><font color=#357EC7>35 µl</font></b></font></b></td></tr></body></table><font color = "#800517"><i>Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.</i></font><br></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for at least <b><font color=#357EC7>1 hr</font></b>.<br><font color = "#800517"><i>Incubation overnight is better.</i></font><br></li></p><p><li><font color = "#800517"><i>Purify digested insert using PCR product purification kit.</i></font><br></li></p><p><li><b><font size=3>Digestion of BioBrick vector: Recommended procedure</font></b><br>Use the following table as a checklist for preparing the reaction in reaction tube (2):<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>BioBrick vector</font></td><td><font color=#357EC7>10X BSA</font></td><td><font color=#357EC7>10X restriction buffer</font></td><td><font color=#357EC7>restriction enzyme1</font></td><td><font color=#357EC7>restriction enzyme2</font></td><td><font color=#357EC7>distilled H2O</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Restriction Digest - vector</font></td><td><b><b><font color=#357EC7>7/C</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></td><td><b><b><font color=#357EC7>0.2 µl</font></b></td><td><b><b><font color=#357EC7>0.2 µl</font></b></td><td><b><font color=#357EC7>Make up the volume to <b><font color=#357EC7>10 µl</font></b></font></b></td></tr></body></table><font color = "#800517"><i>Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.</i></font><br></li></p><p><li>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>12 hrs</font></b>(overnight).<br></li></p><p><li>Measure out <b><font color=#357EC7>0.1 µl</font></b> of <a href="#CIP" ><font color=#357EC7>CIP</font></a> into reaction tube (2).<br>Incubate at <b><font color=#357EC7>37°C</font></b> for <b><font color=#357EC7>1 hr</font></b>.<br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 14 hrs, 0 mins</font></b></p></html>

Current revision

Solutions/reagents:

Parameters:

  • V - Volume of PCR product DNA obtained (µl).
  • C - Concentration of BioBrick vector (µg/µl).

Equipment:

  • Incubator
  • Reaction tubes

Steps:

  1. Digestion of PCR product
    Use the following table as a checklist for preparing the reaction in reaction tube (1):

     PCR product DNA10X BSA10X restriction bufferrestriction enzyme1restriction enzyme2distilled H2O
    Restriction Digest - PCR productV3.5 µl3.5 µl0.2 µl0.2 µlMake up the volume to 35 µl
    Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.
  2. Incubate at 37°C for at least 1 hr.
    Incubation overnight is better.
  3. Purify digested insert using PCR product purification kit.
  4. Digestion of BioBrick vector: Recommended procedure
    Use the following table as a checklist for preparing the reaction in reaction tube (2):

     BioBrick vector10X BSA10X restriction bufferrestriction enzyme1restriction enzyme2distilled H2O
    Restriction Digest - vector7/C1 µl1 µl0.2 µl0.2 µlMake up the volume to 10 µl
    Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.
  5. Incubate at 37°C for 12 hrs(overnight).
  6. Measure out 0.1 µl of CIP into reaction tube (2).
    Incubate at 37°C for 1 hr.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 14 hrs, 0 mins

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