Calgary/protocols

From 2007.igem.org

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<li><a href="#transformation" title="Bacterial Transformation Protocol"> Bacterial Transformation </a></li>
<li><a href="#transformation" title="Bacterial Transformation Protocol"> Bacterial Transformation </a></li>
<li><a href="#rehydration" title="Protocol for rehydrating cells from registery"> Rehydration </a></li>
<li><a href="#rehydration" title="Protocol for rehydrating cells from registery"> Rehydration </a></li>
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<li><a href="#pcr" title="Protocol for pcr"> PCR </a></li>
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<p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="pcr"> PCR Protocol </a> </p>
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<table width="90%" border="1px" style="margin-bottom:15px;">
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  <tr>
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    <td><b>Reagent</b></td>
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    <td><b>Volume ( 1x )</b></td>
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    <td><b>Volume ( 3x )</b></td>
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    <td><b>Volume ( 5x )</b></td>
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    <td><b>Volume ( 15x )</b></td>
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  </tr>
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  <tr>
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    <td>Sterile H2O</td>
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    <td>36 ul</td>
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    <td>108 ul</td>
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    <td>180 ul</td>
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    <td>540 ul</td>
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  </tr>
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  <tr>
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    <td>10X Taq Buffer</td>
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    <td>5 ul</td>
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    <td>15 ul</td>
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    <td>25 ul</td>
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    <td>75 ul</td>
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  </tr>
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  <tr>
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    <td>2mM dNTPs</td>
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    <td>5 ul</td>
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    <td>15 ul</td>
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    <td>25 ul</td>
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    <td>75 ul</td>
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  </tr>
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  <tr>
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    <td>Forward Primer (100 ug/ul) </td>
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    <td>1 ul</td>
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    <td>3 ul</td>
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    <td>5 ul</td>
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    <td>15 ul</td>
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  </tr>
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  <tr>
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    <td>Reverse Primer (100 ug/ul) </td>
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    <td>1 ul</td>
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    <td>3 ul</td>
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    <td>5 ul</td>
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    <td>15 ul</td>
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  </tr>
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  <tr>
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    <td>50mM MgCl2</td>
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    <td>1.5 ul</td>
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    <td>4.5 ul</td>
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    <td>7.5 ul</td>
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    <td>22.5 ul</td>
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  </tr>
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  <tr>
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    <td>Taq Polymerase (50 ug/ul)</td>
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    <td>0.5 ul</td>
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    <td>1.5 ul</td>
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    <td>2.5 ul</td>
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    <td>7.5 ul</td>
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  </tr>
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</table>
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<p><b> Thermocycler Conditions </b></p>
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<ul>
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<li> 1 Cycle - 6 minutes at 95 degrees Celsius </li>
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<li> 36 cycles
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<ul>
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<li> 1 minute at 95 degrees Celsius </li>
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<li> 1 minute at 58 degrees Celsius ( this step done at 65 degrees Celsius for higher GC content ) </li>
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<li> 1 minute at 72 degrees Celsius </li>
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</ul>
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</li>
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<li> 1 Cycle - 10 minutes at 72 degrees Celsius then HOLD at 4 degrees Celsius </li>
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</ul>
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<p> Conditions were varied as needed. For example in cases of longer products all 1 minute times were increased to 1.5 to 3 minutes</p>
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Revision as of 18:58, 12 October 2007

Protocols


Bacterial Transformation

  • Thaw 100 ul of competen cells (per transformation) on ice just before they are needed
  • Add DNA (max 20ul) thawed cells and mix by flicking the side of the tube. Leave on ice for 30 minutes
  • Heat shock for 2 minutes at 42 degrees Celsius or 5 minutes at 37 degrees Celsius
  • Place on Ice for 5 minutes
  • Add 250ul SOC medium to each tube
  • Incubate for 30 to 60 minutes with shaking at 37 degrees Celsius. (Note that for Kanamycin containing plasmides always use one hour)
  • Spin down to remove all supernatant except approximately 100 ul
  • Plate approximately 30 ul on each of two antibiotic plates
  • Grow overnight at 37 degrees Celsius

For this protocol we used three controls

  • Positive Control - pBluescript in TOP10 cells on amp resistant plates
  • Negative Control - TOP10 cells grown on amp resistant plates
  • Negative Control - DB31 cells on amp resistant plates

Rehydration

Biobrick parts are shipped from the registry in a dehydrated from. As such they must be rehydrated before they can be used.

  • Puncture a hole through the foil with a pipette tip into the well that corresponds to the Biobrick - standard part that you want
  • Add 15 ul of diH20 (deionized water)
  • Take 1 ul DNA and transform into your desired competent cells, plate out onto a plate with the correct antibiotic and grow overnight. Your goal here is to obtain single colonies

For this protocol we used three controls

  • Positive Control - pBluescript in TOP10 cells on amp resistant plates
  • Negative Control - TOP10 cells grown on amp resistant plates
  • Negative Control - DB31 cells on amp resistant plates

PCR Protocol

Reagent Volume ( 1x ) Volume ( 3x ) Volume ( 5x ) Volume ( 15x )
Sterile H2O 36 ul 108 ul 180 ul 540 ul
10X Taq Buffer 5 ul 15 ul 25 ul 75 ul
2mM dNTPs 5 ul 15 ul 25 ul 75 ul
Forward Primer (100 ug/ul) 1 ul 3 ul 5 ul 15 ul
Reverse Primer (100 ug/ul) 1 ul 3 ul 5 ul 15 ul
50mM MgCl2 1.5 ul 4.5 ul 7.5 ul 22.5 ul
Taq Polymerase (50 ug/ul) 0.5 ul 1.5 ul 2.5 ul 7.5 ul

Thermocycler Conditions

  • 1 Cycle - 6 minutes at 95 degrees Celsius
  • 36 cycles
    • 1 minute at 95 degrees Celsius
    • 1 minute at 58 degrees Celsius ( this step done at 65 degrees Celsius for higher GC content )
    • 1 minute at 72 degrees Celsius
  • 1 Cycle - 10 minutes at 72 degrees Celsius then HOLD at 4 degrees Celsius

Conditions were varied as needed. For example in cases of longer products all 1 minute times were increased to 1.5 to 3 minutes