Team:Calgary/Lab/Protocol
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<div class="heading"> | <div class="heading"> | ||
+ | <img src="http://i1001.photobucket.com/albums/af132/igemcalgary/l.gif" align="left"> | ||
+ | <br> | ||
Lab Protocols | Lab Protocols | ||
</div> | </div> | ||
- | <div class="desc"> | + | |
- | The following are the protocols that are used in the lab this year. Some modifications may have | + | <div class="desc" width="750"> |
- | + | ||
+ | The following are the protocols that are used in the lab this year. Some modifications may have occurred during this summer. If there are any changes, those changes can be found in the notebook. | ||
<br> | <br> | ||
- | < | + | All team members of this year's iGEM team have successfully completed WHMIS and Biosafety Training. |
- | <table width=" | + | |
+ | <!-- MENU TABLE --> | ||
+ | <table width="700"> | ||
<tr> | <tr> | ||
<td valign="top"><ul id="sub"> | <td valign="top"><ul id="sub"> | ||
- | <li><a href="# | + | <li><a href="#rehydration" title="Protocol for rehydrating plasmid from Registry"> Rehydration </a></li> |
- | <li><a href="# | + | <li><a href="#pcr" title="Colony Polymerase Chain Reaction (PCR)"> Colony PCR </a></li> |
- | <li><a href="#pcr" title=" | + | <li><a href="#pcr" title="Gradient PCR"> Gradient PCR </a></li> |
- | + | <li><a href="#transformation" title="Bacteria Transformation"> Bacterial Transformation </a></li> | |
+ | </ul></td> | ||
<td valign="top"><ul id ="sub"> | <td valign="top"><ul id ="sub"> | ||
<li><a href="#ct" title="Construction Technique"> Construction Technique</a></li> | <li><a href="#ct" title="Construction Technique"> Construction Technique</a></li> | ||
- | <li><a href="#pp" title="Plasmid Prep"> Plasmid Prep </a></li> | + | <li><a href="#pp" title="Plasmid Isolation"> Plasmid Prep </a></li> |
+ | <li><a href="#pp2" title="Plasmid Isolation 2"> Plasmid Prep 2 </a></li> | ||
<li><a href="#ap" title="Antarctic Phosphatase"> Vector Dephosphorylation </a></li> | <li><a href="#ap" title="Antarctic Phosphatase"> Vector Dephosphorylation </a></li> | ||
</ul></td> | </ul></td> | ||
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<li><a href="#overnight" title="overnight"> Over Night Growth </a></li> | <li><a href="#overnight" title="overnight"> Over Night Growth </a></li> | ||
<li><a href="#gsp" title="Glycerol Stock"> Glycerol Stock Preparation </a></li> | <li><a href="#gsp" title="Glycerol Stock"> Glycerol Stock Preparation </a></li> | ||
+ | <li><a href="#pcrp" title="PCR Purification"> PCR Purification </a></li> | ||
</ul></td> | </ul></td> | ||
<td valign="top"><ul id="sub"> | <td valign="top"><ul id="sub"> | ||
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<li><a href="#rd" title="Restriction Digest"> Restriction Digest </a></li> | <li><a href="#rd" title="Restriction Digest"> Restriction Digest </a></li> | ||
<li><a href="#lp" title="Ligation"> Ligation </a></li> | <li><a href="#lp" title="Ligation"> Ligation </a></li> | ||
+ | <li><a href="#mut" title="Quikchange Site Directed Mutagenesis">Site Directed Mutagenesis</a></li> | ||
</ul></td> | </ul></td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
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+ | </td> | ||
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+ | <tr> | ||
+ | <td> | ||
+ | <!--Bacterial Transformation--> | ||
+ | <table width="700"> | ||
<tr> | <tr> | ||
- | <td style="width:85%;">< | + | <td style="width:85%;"><div class="heading"><a name="transformation"></a>Bacterial Transformation</div></td> |
<td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
- | < | + | <ol> |
<li> Thaw 100 μL of competent cells (per transformation) on ice just before they are needed</li> | <li> Thaw 100 μL of competent cells (per transformation) on ice just before they are needed</li> | ||
<li> Add DNA (max 20ul) thawed cells and mix by flicking the side of the tube. Leave on ice for 30 minutes</li> | <li> Add DNA (max 20ul) thawed cells and mix by flicking the side of the tube. Leave on ice for 30 minutes</li> | ||
- | <li> Heat shock | + | <li> Heat shock 5 minutes at 37 degrees Celsius</li> |
- | <li> Place on | + | <li> Place on ice for 5 minutes </li> |
<li> Add 250ul SOC medium to each tube</li> | <li> Add 250ul SOC medium to each tube</li> | ||
- | <li> Incubate for 30 to 60 minutes with shaking at 37 degrees Celsius. (Note that for Kanamycin containing | + | <li> Incubate for 30 to 60 minutes with shaking at 37 degrees Celsius. (Note that for Kanamycin containing plasmids always use one hour)</li> |
<li> Spin down to remove all supernatant except approximately 100 μL</li> | <li> Spin down to remove all supernatant except approximately 100 μL</li> | ||
<li> Plate approximately 30 μL on each of two antibiotic plates </li> | <li> Plate approximately 30 μL on each of two antibiotic plates </li> | ||
<li> Grow overnight at 37 degrees Celsius </li> | <li> Grow overnight at 37 degrees Celsius </li> | ||
- | </ | + | </ol> |
<p> For this protocol we used a couple of controls | <p> For this protocol we used a couple of controls | ||
<ul> | <ul> | ||
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</p> | </p> | ||
</div> | </div> | ||
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<div style="margin-bottom:60px"> | <div style="margin-bottom:60px"> | ||
- | <table width=" | + | <table width="700"> |
<tr> | <tr> | ||
- | <td width="85%">< | + | <td width="85%"><div class="heading"><a style="text-decoration:none" name="rehydration"></a> Rehydration</div></td> |
<td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
<p> Biobrick parts are shipped from the registry in a dehydrated from. As such they must be rehydrated before they can be used. </p> | <p> Biobrick parts are shipped from the registry in a dehydrated from. As such they must be rehydrated before they can be used. </p> | ||
- | < | + | <ol> |
<li> Puncture a hole through the foil with a pipette tip into the well that corresponds to the Biobrick - standard part that you want</li> | <li> Puncture a hole through the foil with a pipette tip into the well that corresponds to the Biobrick - standard part that you want</li> | ||
<li> Add 15 μL of diH20 (deionized water)</li> | <li> Add 15 μL of diH20 (deionized water)</li> | ||
<li> Let the water sit for 5 minutes</li> | <li> Let the water sit for 5 minutes</li> | ||
<li>Take 2 μL 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</li> | <li>Take 2 μL 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</li> | ||
- | </ | + | </ol> |
</div> | </div> | ||
+ | <br> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td width="750" bgcolor="#222222" height="10"> | ||
+ | </td> | ||
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+ | |||
<div style="margin-bottom:60px"> | <div style="margin-bottom:60px"> | ||
- | <table width=" | + | <table width="700"> |
<tr> | <tr> | ||
- | <td width="85%"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="pcr">Taq PCR Protocol </ | + | <td width="85%"><div class="heading"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="pcr"></a>Taq PCR Protocol </p></div></td> |
<td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
- | <table width=" | + | <table width="630" border="1px" style="margin-bottom:15px;"> |
<tr> | <tr> | ||
<td><b>Reagent</b></td> | <td><b>Reagent</b></td> | ||
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</table> | </table> | ||
<p><b> Thermocycler Conditions </b></p> | <p><b> Thermocycler Conditions </b></p> | ||
- | < | + | <ol> |
<li> 1 Cycle - 6 minutes at 95 degrees Celsius </li> | <li> 1 Cycle - 6 minutes at 95 degrees Celsius </li> | ||
<li> 36 cycles of: | <li> 36 cycles of: | ||
- | < | + | <ol> |
<li> 1 minute at 95 degrees Celsius </li> | <li> 1 minute at 95 degrees Celsius </li> | ||
<li> 1 minute at 58 degrees Celsius ( this step done at 65 degrees Celsius for higher GC content ) </li> | <li> 1 minute at 58 degrees Celsius ( this step done at 65 degrees Celsius for higher GC content ) </li> | ||
<li> 1 minute at 72 degrees Celsius </li> | <li> 1 minute at 72 degrees Celsius </li> | ||
- | </ | + | </ol> |
</li> | </li> | ||
<li> 1 Cycle - 10 minutes at 72 degrees Celsius then HOLD at 4 degrees Celsius </li> | <li> 1 Cycle - 10 minutes at 72 degrees Celsius then HOLD at 4 degrees Celsius </li> | ||
- | </ | + | </ol> |
<p> Conditions were varied as needed. For example in cases of longer products all 1 minute times were increased to 1.5 or even 3 minutes</p> | <p> Conditions were varied as needed. For example in cases of longer products all 1 minute times were increased to 1.5 or even 3 minutes</p> | ||
</div> | </div> | ||
+ | <br> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
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+ | <td width="750" bgcolor="#222222" height="10"> | ||
+ | </td> | ||
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<div style="margin-bottom:60px"> | <div style="margin-bottom:60px"> | ||
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<tr> | <tr> | ||
- | <td style="width:85%;">< | + | <td style="width:85%;"><div class="heading"><a style="text-decoration:none" name="pp"></a> Plasmid Preparation Protocol </div></td> |
<td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
- | < | + | The volumes of different solutions may vary depending on the type of kit we used. The main kits that we used this summer was ordered from Sigma and Qiagen |
+ | <ol> | ||
<li><em>Harvest Cells</em> | <li><em>Harvest Cells</em> | ||
<ul> | <ul> | ||
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</ul> | </ul> | ||
</li> | </li> | ||
- | </ | + | </ol> |
</div> | </div> | ||
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+ | </td> | ||
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<div style="margin-bottom:60px"> | <div style="margin-bottom:60px"> | ||
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<tr> | <tr> | ||
- | <td style="width:85%;">< | + | <td style="width:85%;"><div class="heading"><a style="text-decoration:none" name="ct"></a> Construction Technique </div></td> |
<td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | ||
</tr> | </tr> | ||
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<p> You are now done. If you are going to transform this construction product, add all 21μL to a tube of whichever competent bacteria you're using. </p> | <p> You are now done. If you are going to transform this construction product, add all 21μL to a tube of whichever competent bacteria you're using. </p> | ||
</div> | </div> | ||
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+ | </td> | ||
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<div style="margin-bottom:60px"> | <div style="margin-bottom:60px"> | ||
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<tr> | <tr> | ||
- | <td style="width:85%;"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="platePrep"> LB - Agar Plate Preparation Protocol</ | + | <td style="width:85%;"><div class="heading"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="platePrep"></a> LB - Agar Plate Preparation Protocol</p></div></td> |
<td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
- | < | + | <ol> |
<li>Weigh 35g of LB-Agar powder mix per litre of media desired. One litre makes 40-50 plates</li> | <li>Weigh 35g of LB-Agar powder mix per litre of media desired. One litre makes 40-50 plates</li> | ||
<li>Select an appropriate flask; the lab autoclave will cause flasks half full and above to boil over! Use a 2L flasks for up to .5 L of media, a 4 litre flask for up to 1.5L, etc</li> | <li>Select an appropriate flask; the lab autoclave will cause flasks half full and above to boil over! Use a 2L flasks for up to .5 L of media, a 4 litre flask for up to 1.5L, etc</li> | ||
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<li>kanamycin (stock 50mg/ml, final 50ug/ml)</li> | <li>kanamycin (stock 50mg/ml, final 50ug/ml)</li> | ||
<li>chloramphenicol (stock 50mg/ml, final 10ug/ml)</li> | <li>chloramphenicol (stock 50mg/ml, final 10ug/ml)</li> | ||
- | |||
<li>To achieve final concentrations, add 1mL of stock per 1L of media, except for chloramphenicol, where 0.6mL per 1L of media is added instead</li> | <li>To achieve final concentrations, add 1mL of stock per 1L of media, except for chloramphenicol, where 0.6mL per 1L of media is added instead</li> | ||
</ul> | </ul> | ||
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<li>Pour directly from the flask into sterile petri plates. Use a quick pass with a bunsen burner flame to snuff out bubbles that form during pouring. Do not subject the plate to continuous heat or the plate will melt, and the heat sensitive ingredients added in the previous step will be destroyed. Bubbles can allow cells to access nutrients without being exposed to the plate's antibiotic, and should be blown out immediately before the gel can set. It's a good idea for one person to pour while another flames bubbles. </li> | <li>Pour directly from the flask into sterile petri plates. Use a quick pass with a bunsen burner flame to snuff out bubbles that form during pouring. Do not subject the plate to continuous heat or the plate will melt, and the heat sensitive ingredients added in the previous step will be destroyed. Bubbles can allow cells to access nutrients without being exposed to the plate's antibiotic, and should be blown out immediately before the gel can set. It's a good idea for one person to pour while another flames bubbles. </li> | ||
<li>Allow the plates to stand right side up overnight, or until the gel sets if they are needed sooner. Plates should be stored upside down to keep condensation from falling on the media. Store petri plates in the plastic bags they ship in, in the 4 degree cold room. </li> | <li>Allow the plates to stand right side up overnight, or until the gel sets if they are needed sooner. Plates should be stored upside down to keep condensation from falling on the media. Store petri plates in the plastic bags they ship in, in the 4 degree cold room. </li> | ||
- | </ | + | </ol> |
</div> | </div> | ||
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<div style="margin-bottom:60px"> | <div style="margin-bottom:60px"> | ||
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<tr> | <tr> | ||
- | <td style="width:85%;"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="overnight"> Over Night Growth</ | + | <td style="width:85%;"><div class="heading"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="overnight"></a> Over Night Growth </p></div></td> |
<td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | ||
</tr> | </tr> | ||
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<li> 10mL culture tube. Use 16mm x 160mm or 16mm x 125mm</li> | <li> 10mL culture tube. Use 16mm x 160mm or 16mm x 125mm</li> | ||
<li> 5 mL LB</li> | <li> 5 mL LB</li> | ||
- | <li> 5 uL 1000X | + | <li> 5 uL 1000X antibiotics</li> |
<li> Single colonies on a plate (best not to start an over night from a glycerol stock)</li> | <li> Single colonies on a plate (best not to start an over night from a glycerol stock)</li> | ||
</ul> | </ul> | ||
</p> | </p> | ||
<p> Protocol </p> | <p> Protocol </p> | ||
- | < | + | <ol> |
<li> Pipet 5uL 1000X antibiotic into culture tube </li> | <li> Pipet 5uL 1000X antibiotic into culture tube </li> | ||
<li> Add 5mL non-contaminated LB. Do this first. Then add antibiotic</li> | <li> Add 5mL non-contaminated LB. Do this first. Then add antibiotic</li> | ||
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<li> Place toothpick or loop in culture tube and stir </li> | <li> Place toothpick or loop in culture tube and stir </li> | ||
<li> Remove toothpick or loop and place culture tube in incubator at 37 C overnight shaking vigorously (250 RPM)</li> | <li> Remove toothpick or loop and place culture tube in incubator at 37 C overnight shaking vigorously (250 RPM)</li> | ||
- | </ | + | </ol> |
</div> | </div> | ||
+ | <br> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
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<div style="margin-bottom:60px"> | <div style="margin-bottom:60px"> | ||
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<tr> | <tr> | ||
- | <td style="width:85%;"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="gsp"> Glycerol Stock Preparation</ | + | <td style="width:85%;"><div class="heading"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="gsp"></a> Glycerol Stock Preparation </p></div></td> |
<td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | ||
</tr> | </tr> | ||
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</ul> | </ul> | ||
<p> Protocol </p> | <p> Protocol </p> | ||
- | < | + | <ol> |
<li> Pipet 0.5mL of 50% glycerol into 3 1.5 screw cap tubes</li> | <li> Pipet 0.5mL of 50% glycerol into 3 1.5 screw cap tubes</li> | ||
<li> Add 0.5mL of overnight culture to each tube </li> | <li> Add 0.5mL of overnight culture to each tube </li> | ||
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<li> Flash freeze in liquid N2 or dry ice/ethanol bath </li> | <li> Flash freeze in liquid N2 or dry ice/ethanol bath </li> | ||
<li> Place in -80 C freezer when frozen </li> | <li> Place in -80 C freezer when frozen </li> | ||
- | </ | + | </ol> |
</div> | </div> | ||
+ | <br> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
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<div style="margin-bottom:60px"> | <div style="margin-bottom:60px"> | ||
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<tr> | <tr> | ||
- | <td style="width:85%;"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="age"> Agarose Gel Electrophoresis Protocol</ | + | <td style="width:85%;"><div class="heading"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="age"></a> Agarose Gel Electrophoresis Protocol </p></div></td> |
<td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | ||
</tr> | </tr> | ||
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</ul> | </ul> | ||
<p> Protocol </p> | <p> Protocol </p> | ||
- | < | + | <ol> |
<li> Measure out 120mL of buffer </li> | <li> Measure out 120mL of buffer </li> | ||
<li> Transfer buffer to 125 mL flask </li> | <li> Transfer buffer to 125 mL flask </li> | ||
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<li> Run the apparatus at 100V for 30 - 45 minutes (make sure to watch that the dye does not run off the gel)</li> | <li> Run the apparatus at 100V for 30 - 45 minutes (make sure to watch that the dye does not run off the gel)</li> | ||
<li> Visualize the gel and record the results</li> | <li> Visualize the gel and record the results</li> | ||
- | </ | + | </ol> |
</div> | </div> | ||
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+ | |||
<div style="margin-bottom:60px"> | <div style="margin-bottom:60px"> | ||
- | <table width=" | + | <table width="700"> |
<tr> | <tr> | ||
- | <td style="width:85%;"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="rd"> | + | <td style="width:85%;"><div class="heading"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="rd"></a> Restriction Digest</p></div></td> |
<td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
- | <p> This protocol is also described as a part of our <a href="#ct" title="construction technique"> Construction Technique</a>. Start by selecting the order of the two parts you will be putting together; the one in front will be referred to as the insert, while the one behind will be referred to as the vector. Both the vector and the insert need to have their own separate tube, at least in the beginning. This is important because it allows for clean addition new parts to a the | + | <p> This protocol is also described as a part of our <a href="#ct" title="construction technique"> Construction Technique</a>. Start by selecting the order of the two parts you will be putting together; the one in front will be referred to as the insert, while the one behind will be referred to as the vector. Both the vector and the insert need to have their own separate tube, at least in the beginning. This is important because it allows for clean addition new parts to a the circuit </p> |
<p> In the Insert Tube... | <p> In the Insert Tube... | ||
<ul> | <ul> | ||
Line 464: | Line 589: | ||
Take the insert out, and put it in a -20°C freezer. </p> | Take the insert out, and put it in a -20°C freezer. </p> | ||
</div> | </div> | ||
+ | <br> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td width="750" bgcolor="#222222" height="10"> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td> | ||
+ | |||
<div style="margin-bottom:60px"> | <div style="margin-bottom:60px"> | ||
- | <table width=" | + | <table width="700"> |
<tr> | <tr> | ||
- | <td style="width:85%;"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="lp"> | + | <td style="width:85%;"><div class="heading"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="lp"></a> Ligation Protocol </p></div></td> |
<td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
<p> This protocol is also described as a part of our <a href="#ct" title="construction technique"> Construction Technique</a>. Start by selecting the order of the two parts you will be putting together; the one in front will be referred to as the insert, while the one behind will be referred to as the vector. Both the vector and the insert need to have their own separate tube, at least in the beginning. This is important because it allows for clean addition new parts to a the circut </p> | <p> This protocol is also described as a part of our <a href="#ct" title="construction technique"> Construction Technique</a>. Start by selecting the order of the two parts you will be putting together; the one in front will be referred to as the insert, while the one behind will be referred to as the vector. Both the vector and the insert need to have their own separate tube, at least in the beginning. This is important because it allows for clean addition new parts to a the circut </p> | ||
- | < | + | <ol> |
<li>Take insert out of the freezer and ad 5 uL of insert and 5 uL f vector to a new tube </li> | <li>Take insert out of the freezer and ad 5 uL of insert and 5 uL f vector to a new tube </li> | ||
<li>Clearly label the remaining tubes of each (insert and vector) as Unligated, put the date on the tube and place in -20 C freezer in case the transformation does not work</li> | <li>Clearly label the remaining tubes of each (insert and vector) as Unligated, put the date on the tube and place in -20 C freezer in case the transformation does not work</li> | ||
<li>To the single tube containing both insert and vector add 10 uL of 2x Quick Ligase Buffer and 1 uL of Quick Ligase.</li> | <li>To the single tube containing both insert and vector add 10 uL of 2x Quick Ligase Buffer and 1 uL of Quick Ligase.</li> | ||
<li>Let this sit at room temperature for 5 minutes</li> | <li>Let this sit at room temperature for 5 minutes</li> | ||
- | </ul> | + | </ol> |
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td width="750" bgcolor="#222222" height="10"> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td> | ||
+ | <div style="margin-bottom:60px"> | ||
+ | <table width="700"> | ||
+ | <tr> | ||
+ | <td width="85%"><div class="heading"><a style="text-decoration:none" name="pp2"></a> Plasmid Preparation/Isolation (For Higher Concentration of Plasmids)</div></td> | ||
+ | <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>This protocol is used to obtain higher concentrations of plasmids.</p> | ||
+ | <ol> | ||
+ | <li>Pick a single colony and inoculate a starter culture of 2-5 mL LB broth | ||
+ | + antibiotic and shake for 8 hours at 37 degrees Celsius</li> | ||
+ | <li>Dilute the starter culture by 1/500 to 1/1000</li> | ||
+ | <li>Isolate the cells by spinning at 3200 rpm for 25 mins holding the | ||
+ | temperature at 4 degrees Celcius.</li> | ||
+ | <li>Resuspend the bacterial pellet with 10mL of buffer P1</li> | ||
+ | <li>Add 10 mL of Buffer P2 and invert the tube several times and incubate | ||
+ | at room temperature for no longer than 5 mins. This step is for lysis of | ||
+ | cells.</li> | ||
+ | <li>Add 10 mL of cold Buffer P3 to the lysate and invert several times.</li> | ||
+ | This step is used to neutralize the lysis reaction.</li> | ||
+ | <li>Pour the neutralized lysate through the barrel of the QIAfilter</li> | ||
+ | Cartridge (included in kit) for 10 mins. Let this sit so that the later of | ||
+ | proteins, genomic DNA, and detergent will float on top of the solution</li> | ||
+ | <li>Remove the cap from the cartridge outlet nozzle. Gently insert the | ||
+ | plunger into the cartridge and filter the lysate into a 50 mL centrifuge | ||
+ | tube.</li> | ||
+ | <li>Add 2.5 Ml of Buffer ER to the filter lysate, mix by inverting and then | ||
+ | incubate for 30 mins in ice</li> | ||
+ | <li> Equilibrate a Qiagen-tip 500 by adding 10 ml of Buffer QBT and allow | ||
+ | gravity to empty the solution.</li> | ||
+ | <li> Pour the lysate through the filter and allow the solution to flow | ||
+ | through via gravity flow.</li> | ||
+ | <li> Wash the tip with Buffer QC (30 mL) twice.</li> | ||
+ | <li> Elute the DNA with 15mL of Buffer QN</li> | ||
+ | <li> Precipitate the DNA by adding 0.7 volumes room temperature isopropanol | ||
+ | to the eluted DNA. Centrifuge for 90 mins at 3000 rpm.</li> | ||
+ | <li> Empty the supernatant slowly and carefully, the pellet is hard to see.</li> | ||
+ | <li>Wash the pellet with 5mL ethanol (96-100%) and centrifuge for 90 mins | ||
+ | at 3000 rpm. Carefully decant the supernatant without disturbing the | ||
+ | pellet.</li> | ||
+ | <li> Air dry the pellet for 10 mins and then redissolve the plasmid in | ||
+ | buffer TE with a suitable volume.</li> | ||
+ | |||
+ | </ol> | ||
+ | </div> | ||
+ | <br> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td width="750" bgcolor="#222222" height="10"> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td> | ||
+ | <div style="margin-bottom:60px"> | ||
+ | <table width="700"> | ||
+ | <tr> | ||
+ | <td width="85%"><div class="heading"><a style="text-decoration:none" name="pcrp"></a> PCR Purification</div></td> | ||
+ | <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>The kit we used over the summer was purchased from Qiagen. This kit is | ||
+ | used to purify the PCR product. | ||
+ | </p> | ||
+ | <ol> | ||
+ | <li>Add 5 volumes of Buffer PB to 1 volume of PCR product and then mix.</li> | ||
+ | <li> Place a spin column in a 2 mL collection tube.</li> | ||
+ | <li> Apply the mixture of PB buffer + PCR product through the QIA column and | ||
+ | centrifuge for 1 minute.</li> | ||
+ | <li> Discard the flow-through and place the column back in the tube.</li> | ||
+ | <li> Wash the product using 0.75 mL buffer PE and then centrifuge for 1 min.</li> | ||
+ | <li> Discard the flow-through and place the column back into the tube.</li> | ||
+ | Centrifuge the column inside the tube to discard the additional fluid in | ||
+ | the column.</li> | ||
+ | <li> Elute the DNA into a clean microcentrifuge tube with buffer EB. | ||
+ | </li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | <br> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td width="750" bgcolor="#222222" height="10"> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td> | ||
+ | <div style="margin-bottom:60px"> | ||
+ | <table width="700"> | ||
+ | <tr> | ||
+ | <td width="85%"><div class="heading"><a style="text-decoration:none" name="mut"></a> Quikchange Site Directed Mutagenesis</div></td> | ||
+ | <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p> The kit that we used was purchased from Stratagene.</p> | ||
+ | <ol> | ||
+ | <li>Design and synthesis two complimentary oligonucleotides containing the mutation.</li> | ||
+ | <li>Prepare the control + sample reaction | ||
+ | </li> | ||
+ | <br> | ||
+ | Control: | ||
+ | <ul> | ||
+ | <li>5 µL of 10x reaction buffer</li> | ||
+ | <li>2 µl (10ng) of pWhitescript 4.5kb control plasmid</li> | ||
+ | <li>1.25 µL (125 ng) of control primer 1</li> | ||
+ | <li>1.25 µL (125 ng) of control primer 2</li> | ||
+ | <li>1 µL of dNTP mix</li> | ||
+ | <li>3 µL of QuikSolution</li> | ||
+ | <li>36.5 µL double distilled water to a final volume of 50 µL</li> | ||
+ | <li>+ 1 µL of PfuTurbo DNA polymerase</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | Sample: | ||
+ | <ul> | ||
+ | <li>5 µL of 10X reaction buffer</li> | ||
+ | <li>X µL (10ng) of dsDNA template</li> | ||
+ | <li>X µL (125ng) forward primer</li> | ||
+ | <li>X µL (125ng) reverse primer</li> | ||
+ | <li>1 µL of dNTP mix</li> | ||
+ | <li>3 µL of QuikSolution</li> | ||
+ | <li>ddH20 to a final solution of 50 µL</li> | ||
+ | <li>+ 1 µL of Pfu Turbo DNA polymerase</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | <li> | ||
+ | Follow the cycling parameters: | ||
+ | <ul> | ||
+ | <li>Segment 1: 1 cycle (95 degrees Celsius, 1 minute)</li> | ||
+ | <li>Segment 2: 18 cycles (95 degrees Celsius, 50 secs; 60 degrees Celsius, 50 | ||
+ | secs; 68 degrees Celsius, 1 min/kb of plasmid length)</li> | ||
+ | <li>Segment 3: 1 cycle (68 degrees Celsius, 7 minutes)</li> | ||
+ | </ul> | ||
+ | <br> | ||
+ | <li>Add 1 µL of DpnI restriction enzyme to the amplified reaction. Mix | ||
+ | gently by pipeting the solution up and down and then incubate at 37 | ||
+ | degrees Celsius for 1 hour.</li> | ||
+ | <li>Thaw the XL-10 Gold ultracompetent cells on ice. Add 45 µL to a | ||
+ | prechilled 14 mL BD Falcon polypropylene round-bottom tube.</li> | ||
+ | <li>Add 2 µL of the â=-ME mix to the 45 µL of cells</li> | ||
+ | <li>Swirl content and incubate cells on ice for 10 mins and gently swirling | ||
+ | every 2 mins.</li> | ||
+ | <li>Transfer 2 µL of the DpnI treated DNA to the cells</li> | ||
+ | <li>Preheat NZY+ broth in a 42 degrees Celsius water bath.</li> | ||
+ | <li> Heat shock cells in a 42 degrees Celsius water bath for 30 seconds</li> | ||
+ | <li>Incubate the tubes on ice for 2 minutes</li> | ||
+ | <li> Add 500 µL of preheated NZY+ broth to each tube then incubate the tube | ||
+ | at 37 degrees Celsius for 1 hour shaking at 225-250 rpm.</li> | ||
+ | <li>For the sample, plate 250 µL of cells on appropriate plate. For the | ||
+ | control, plate 250 µL of cells on the plate. Before plating, X-gal and | ||
+ | IPTG is required for the controls.</li> | ||
+ | <li>Incubate for 37 degrees Celsius for +16 hours.</li> | ||
+ | |||
+ | |||
+ | </ol> | ||
+ | </div> | ||
+ | <br> | ||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | |||
+ | </table> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
</div> | </div> | ||
</body> | </body> | ||
</html> | </html> |
Latest revision as of 21:14, 2 October 2009
UNIVERSITY OF CALGARY