Difference between revisions of "Team:SJTU-BioX-Shanghai/Protocol"
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| − | + | </div> | |
| − | < | + | <div class="big-head h1-my-responsive"> <img src="https://static.igem.org/mediawiki/2017/5/52/T--SJTU-BioX-Shanghai--quan.png" class="img-fluid">Protocol</div> |
| − | + | <div class="container maink"> | |
| − | + | <div class="container maintext"> | |
| − | + | <div class="row"> | |
| − | + | <div class="scrcol col-lg-3"> | |
| − | + | <div class="scroll-box grey lighten-5"> | |
| − | + | <nav class="list-unstyled" id="navbar-left" role="navigation"> | |
| + | |||
| + | <li class="nav-item"> | ||
| + | <a class="nav-link" href="#section1">PCR</a> | ||
| + | </li> | ||
| + | <li class="nav-item"> | ||
| + | <a class="nav-link" href="#section2">Restriction Digestion</a> | ||
| + | </li> | ||
| + | <li class="nav-item"> | ||
| + | <a class="nav-link" href="#section3">Ligation</a> | ||
| + | </li> | ||
| + | <li class="nav-item"> | ||
| + | <a class="nav-link" href="#section4">DNA Gel Electrophoresis</a> | ||
| + | </li> | ||
| + | <li class="nav-item"> | ||
| + | <a class="nav-link" href="#section5">Transformation</a> | ||
| + | </li> | ||
| + | <li class="nav-item"> | ||
| + | <a class="nav-link" href="#section6">Purification and Gel Extraction & Plasmid Extraction</a> | ||
| + | </li> | ||
| + | <li class="nav-item"> | ||
| + | <a class="nav-link" href="#section7">Cotransformation</a> | ||
| + | </li> | ||
| + | <li class="nav-item"> | ||
| + | <a class="nav-link" href="#section8">Characterization</a> | ||
| + | </li> | ||
| + | </nav> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | <div class="col-12 col-lg-9"> | |
| − | + | <div class="my-title h5-my-responsive" id="section1">PCR</div> | |
| − | + | <p class="my-title2">Materials:</p> | |
| − | < | + | <p>Buffer</br>Mg<sup>2+</sup></br>dNTP</br>Template</br>F/R-primer</br>DNA enzyme</p> |
| − | < | + | <p class="my-title2">Methods:</p> |
| − | < | + | <ol type="1" start="1"> |
| − | </ | + | <li>Combination of all reagents</li> |
| + | </ol> | ||
| + | <div class="figure-intro"> | ||
| + | <table class="table table-res table-res2 table-hover" align="center"> | ||
| + | |||
| + | <thead> | ||
| + | <tr> | ||
| + | <td>Name of reagent</td> | ||
| + | <td>volume (μl)</td> | ||
| + | </tr> | ||
| + | </thead> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td>Buffer</td> | ||
| + | <td>2</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Mg<sup>2+</sup></td> | ||
| + | <td>2</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Template</td> | ||
| + | <td>20 ng for plasmid or 200 ng for genome</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>F/R-primer</td> | ||
| + | <td>0.6 for each</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Enzyme</td> | ||
| + | <td>0.4</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Sterile Water</td> | ||
| + | <td>make reaction up to 20μ in total</td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
</div> | </div> | ||
| − | <div class=" | + | <ol type="1" start="2"> |
| − | < | + | <li> Set the PCR machine to run the following steps:</li> |
| − | < | + | </ol> |
| − | </ | + | <div class="figure-intro"> |
| + | <table class="table table-res table-res2 table-hover" align="center"> | ||
| + | |||
| + | <thead> | ||
| + | <tr> | ||
| + | <td>Stage</td> | ||
| + | <td>Temperature (℃)</td> | ||
| + | <td>Time</td> | ||
| + | </tr> | ||
| + | </thead> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td>initial denaturation</td> | ||
| + | <td>94</td> | ||
| + | <td>2 min</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> </td> | ||
| + | <td>94 (denaturation)</td> | ||
| + | <td>15 seconds</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>30 cycles</td> | ||
| + | <td>40-55 (annealing)</td> | ||
| + | <td>30 seconds</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> </td> | ||
| + | <td>68 (extension)</td> | ||
| + | <td>30 seconds per bp</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Final extension</td> | ||
| + | <td>68</td> | ||
| + | <td>7 min</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Incubate</td> | ||
| + | <td>12</td> | ||
| + | <td>Infinite</td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
</div> | </div> | ||
| − | <div class=" | + | |
| − | < | + | <div class="my-title h5-my-responsive" id="section2">Restriction Digestion</div> |
| − | < | + | <p class="my-title2">Materials:</p> |
| − | </ | + | <p>Restriction Enzyme: Fisher FastDigest Restriction Enzyme</br>Green Buffer</br>DNA samplesr</br>Sterile Water</p> |
| + | <p class="my-title2">Methods:</p> | ||
| + | <p>Note: the components listed below are for double digestion!</p> | ||
| + | <div class="figure-intro"> | ||
| + | <table class="table table-res table-res2 table-hover" align="center"> | ||
| + | <thead> | ||
| + | <tr> | ||
| + | <td>Component</td> | ||
| + | <td>volume (μl)</td> | ||
| + | </tr> | ||
| + | </thead> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td>Green Buffer</td> | ||
| + | <td>2</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>DNA sample</td> | ||
| + | <td>600 ng for plasmid or up to 200 ng for PCR production</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Template</td> | ||
| + | <td>20 ng for plasmid or 200 ng for genome</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Restriction Enzyme</td> | ||
| + | <td>1 for each</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Sterile Water</td> | ||
| + | <td>make reaction up to 20μ in total</td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
</div> | </div> | ||
| − | <div class=" | + | <p>Set up the reaction following the above table and incubate at 37℃ for 15-30 min and do the gel electrophoresis or purification later</p> |
| − | < | + | <div class="my-title h5-my-responsive" id="section3">Ligation</div> |
| − | < | + | <p class="my-title2">Materials:</p> |
| − | </ | + | <p>T4 DNA Ligase</br>T4 DNA Ligase Buffer</br>Vector Plasmid</br>Insert DNA</br>Sterile Water</p> |
| + | <p class="my-title2">Methods:</p> | ||
| + | <div class="figure-intro"> | ||
| + | <table class="table table-res table-res2 table-hover" align="center"> | ||
| + | <thead> | ||
| + | <tr> | ||
| + | <td>Component</td> | ||
| + | <td>volume or mass</td> | ||
| + | </tr> | ||
| + | </thead> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td>T4 DNA Ligase Buffer</td> | ||
| + | <td>2 μ</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Vector Plasmid</td> | ||
| + | <td>50 ng</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Insert DNA</td> | ||
| + | <td>require molar ratio of 3:1 to 7:1 (insert:vector)</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>T4 DNA Ligase</td> | ||
| + | <td>0.2 μ</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Sterile Water</td> | ||
| + | <td>make reaction up to 20μ in total</td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
</div> | </div> | ||
| + | <p>Make the reaction and incubate at room temperature for 4-6 hours</p> | ||
| + | <p>Note: for how to calculate volumes of vector and insert DNA, we highly recommend <a target="_blank" href="http://nebiocalculator.neb.com/#!/ligation">NEBbioCalculator</a></p> | ||
| + | <p>Sterile Water</p> | ||
| + | <div class="my-title h5-my-responsive" id="section4">DNA Gel Electrophoresis</div> | ||
| + | <p class="my-title2">Materials:</p> | ||
| + | <p>Agarose</br>TBE Buffer</br>Gel board</br>EB</br>DNA×10 or×6 Loading Buffer</br>Electrophoresis tank</br>sample</br>marker 2000 or 15000</p> | ||
| + | |||
| + | <p class="my-title2">Methods:</p> | ||
| + | <ol type="1" start="1"> | ||
| + | <li>Make a 1.0%-1.5% Agarose gel according to the volume of your gel board (eg. add 0.45g Agarose in 30ml TBE if choose a concentration of 1.5%)</li> | ||
| + | <li>Heat the container with the mixture until it is complete dissolved. When the solution starts boiling, stop heating, shake the container until materials mix well and heat again until the mixture become clear</li> | ||
| + | <li>Wait until the solution cools down to 50℃,add 0.01% EB (eg. 3μin 30ml) or non-poisonous dyes instead</li> | ||
| + | <li>Pour the solution into a gel board and insert the comb</li> | ||
| + | <li>Set the gel at room temperature and wait until it completely solidifies</li> | ||
| + | <li>Remove the comb and transfer the gel to the electrophoresis tank</li> | ||
| + | <li>Add samples into pores with 40% DNA Loading Buffer (eg. 2μLoading Buffer in 5μDNA sample)</li> | ||
| + | <li>Run the gel for 20 min at 100V, 100mA</li> | ||
| + | </ol> | ||
| + | |||
| + | <div class="my-title h5-my-responsive" id="section5">Transformation</div> | ||
| + | <p class="my-title2">Materials:</p> | ||
| + | <p>LB broth</br>Ice</br>Selection plates with corresponding antibiotics.</p> | ||
| + | <p class="my-title2">Methods:</p> | ||
| + | <ol type="1" start="1"> | ||
| + | <li>Put 50µL competent E. coli cells on ice for 10 minutes</li> | ||
| + | <li>Add 20 µl DNA from a ligation reaction mix or 10-100 ng plasmid </li> | ||
| + | <li>Incubate the mixture on ice for 30 minutes</li> | ||
| + | <li>Heat shock at exactly 42°C for exactly 45 seconds</li> | ||
| + | <li>Place on ice for 3 minutes</li> | ||
| + | <li>add the mixture into 500 µL LB broth</li> | ||
| + | <li>Incubate at 37°C and 200-250 rpm for one hour</li> | ||
| + | <li>For ligation reaction DNA: 500 µl of each transformation reaction onto a selection plate. | ||
| + | For known plasmid: 100-200 µL of each transformation reaction onto a selection plate | ||
| + | </li> | ||
| + | <li>Incubate overnight at 37°C with plates upside down.</li> | ||
| + | </ol> | ||
| + | <div class="my-title h5-my-responsive" id="section6">Purification and Gel Extraction & Plasmid Extraction</div> | ||
| + | <p><strong>Purification </strong>of PCR or restriction products was performed according to the OMEGA Cycle Pure Kit D6492.</p> | ||
| + | <p><strong>Gel Extraction </strong>was performed according to the OMEGA Gel Extraction Kit D2500.</p> | ||
| + | <p><strong>Plasmid Extraction</strong> was performed according to the BIOMIGA Plasmid Miniprep Kit.</p> | ||
| + | <div class="my-title h5-my-responsive" id="section7">Cotransformation</div> | ||
| + | <div class="my-title h5-my-responsive" id="section8">Characterization</div> | ||
| + | |||
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Revision as of 03:43, 30 October 2017
ProtocolPCR
Materials:
BufferMg2+dNTPTemplateF/R-primerDNA enzyme
Methods:
- Combination of all reagents
| Name of reagent | volume (μl) |
| Buffer | 2 |
| Mg2+ | 2 |
| Template | 20 ng for plasmid or 200 ng for genome |
| F/R-primer | 0.6 for each |
| Enzyme | 0.4 |
| Sterile Water | make reaction up to 20μ in total |
- Set the PCR machine to run the following steps:
| Stage | Temperature (℃) | Time |
| initial denaturation | 94 | 2 min |
| 94 (denaturation) | 15 seconds | |
| 30 cycles | 40-55 (annealing) | 30 seconds |
| 68 (extension) | 30 seconds per bp | |
| Final extension | 68 | 7 min |
| Incubate | 12 | Infinite |
Restriction Digestion
Materials:
Restriction Enzyme: Fisher FastDigest Restriction EnzymeGreen BufferDNA samplesrSterile Water
Methods:
Note: the components listed below are for double digestion!
| Component | volume (μl) |
| Green Buffer | 2 |
| DNA sample | 600 ng for plasmid or up to 200 ng for PCR production |
| Template | 20 ng for plasmid or 200 ng for genome |
| Restriction Enzyme | 1 for each |
| Sterile Water | make reaction up to 20μ in total |
Set up the reaction following the above table and incubate at 37℃ for 15-30 min and do the gel electrophoresis or purification later
Ligation
Materials:
T4 DNA LigaseT4 DNA Ligase BufferVector PlasmidInsert DNASterile Water
Methods:
| Component | volume or mass |
| T4 DNA Ligase Buffer | 2 μ |
| Vector Plasmid | 50 ng |
| Insert DNA | require molar ratio of 3:1 to 7:1 (insert:vector) |
| T4 DNA Ligase | 0.2 μ |
| Sterile Water | make reaction up to 20μ in total |
Make the reaction and incubate at room temperature for 4-6 hours
Note: for how to calculate volumes of vector and insert DNA, we highly recommend NEBbioCalculator
Sterile Water
DNA Gel Electrophoresis
Materials:
AgaroseTBE BufferGel boardEBDNA×10 or×6 Loading BufferElectrophoresis tanksamplemarker 2000 or 15000
Methods:
- Make a 1.0%-1.5% Agarose gel according to the volume of your gel board (eg. add 0.45g Agarose in 30ml TBE if choose a concentration of 1.5%)
- Heat the container with the mixture until it is complete dissolved. When the solution starts boiling, stop heating, shake the container until materials mix well and heat again until the mixture become clear
- Wait until the solution cools down to 50℃,add 0.01% EB (eg. 3μin 30ml) or non-poisonous dyes instead
- Pour the solution into a gel board and insert the comb
- Set the gel at room temperature and wait until it completely solidifies
- Remove the comb and transfer the gel to the electrophoresis tank
- Add samples into pores with 40% DNA Loading Buffer (eg. 2μLoading Buffer in 5μDNA sample)
- Run the gel for 20 min at 100V, 100mA
Transformation
Materials:
LB brothIceSelection plates with corresponding antibiotics.
Methods:
- Put 50µL competent E. coli cells on ice for 10 minutes
- Add 20 µl DNA from a ligation reaction mix or 10-100 ng plasmid
- Incubate the mixture on ice for 30 minutes
- Heat shock at exactly 42°C for exactly 45 seconds
- Place on ice for 3 minutes
- add the mixture into 500 µL LB broth
- Incubate at 37°C and 200-250 rpm for one hour
- For ligation reaction DNA: 500 µl of each transformation reaction onto a selection plate. For known plasmid: 100-200 µL of each transformation reaction onto a selection plate
- Incubate overnight at 37°C with plates upside down.
Purification and Gel Extraction & Plasmid Extraction
Purification of PCR or restriction products was performed according to the OMEGA Cycle Pure Kit D6492.
Gel Extraction was performed according to the OMEGA Gel Extraction Kit D2500.
Plasmid Extraction was performed according to the BIOMIGA Plasmid Miniprep Kit.
Cotransformation
Characterization
