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| | <article class="content"> | | <article class="content"> |
| - | <h1>Notebook: Protocol</h1> | + | <h1>Parts</h1> |
| - | <section> | + | |
| - | <p style="text-align: right;">Return to: <a href="https://2014.igem.org/Team:Tsinghua/Notebook/Protocol">Notebook -> Protocol</a></p>
| + | |
| - | <h2>Molecular Cloning</h2>
| + | |
| - | <h3> </h3>
| + | |
| - | <h3>Polymerase Chain Reaction (PCR)</h3>
| + | |
| - | <p>1. The master mix for reactions with FastPfu DNA polymerase contained:</p>
| + | |
| - | <div align="center"><table style="margin-left:50px" border="1" cellspacing="0" cellpadding="0" width="0">
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">Reagent</p></td>
| + | |
| - | <td width="0"><p align="left">Volume</p></td>
| + | |
| - | <td width="0"><p align="left">Final Concentration</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">5x FastPfu Buffer</p></td>
| + | |
| - | <td width="0"><p align="left">10 ul</p></td>
| + | |
| - | <td width="0"><p align="left">1X</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">10 mM</p></td>
| + | |
| - | <td width="0"><p align="left">1 ul</p></td>
| + | |
| - | <td width="0"><p align="left">0.2 mM</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">Primer 1 (25 pmol/ul)</p></td>
| + | |
| - | <td width="0"><p align="left">1 ul</p></td>
| + | |
| - | <td width="0"><p align="left">0.5 pmol/ul</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">Primer 2 (25 pmol/ul)</p></td>
| + | |
| - | <td width="0"><p align="left">1 ul</p></td>
| + | |
| - | <td width="0"><p align="left">0.5 pmol/ul</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">FastPfu (5U/ul)</p></td>
| + | |
| - | <td width="0"><p align="left">0.4 ul</p></td>
| + | |
| - | <td width="0"><p align="left">2U/50ul</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">Template DNA</p></td>
| + | |
| - | <td width="0"><p align="left">variable</p></td>
| + | |
| - | <td width="0"><p align="left">50 pg – 1 ug</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">ddH2O</p></td>
| + | |
| - | <td width="0"><p align="left">To 50 ul</p></td>
| + | |
| - | <td width="0"></td>
| + | |
| - | </tr>
| + | |
| - | </table></div>
| + | |
| - | <p>2. The master mix for reactions with Phusion DNA polymerase contained:</p>
| + | |
| - | <div align="center"><table style="margin-left:50px" border="1" cellspacing="0" cellpadding="0" width="0">
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">Reagent</p></td>
| + | |
| - | <td width="0"><p align="left">Volume</p></td>
| + | |
| - | <td width="0"><p align="left">Final Concentration</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">Phusion DNA Polymerase</p></td>
| + | |
| - | <td width="0"><p align="left">0.5 ul</p></td>
| + | |
| - | <td width="0"><p align="left">1.0 units/50 ul PCR</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">DMSO (optional)</p></td>
| + | |
| - | <td width="0"><p align="left">(1.5 ul)</p></td>
| + | |
| - | <td width="0"><p align="left">3%</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">Template DNA</p></td>
| + | |
| - | <td width="0"><p align="left">variable</p></td>
| + | |
| - | <td width="0"><p align="left">250 ng</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">10 uM Forward Primer</p></td>
| + | |
| - | <td width="0"><p align="left">2.5 ul</p></td>
| + | |
| - | <td width="0"><p align="left">0.5 uM</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">10 uM Reverse Primer</p></td>
| + | |
| - | <td width="0"><p align="left">2.5 ul</p></td>
| + | |
| - | <td width="0"><p align="left">0.5 uM</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">10 mM dNTPs</p></td>
| + | |
| - | <td width="0"><p align="left">1 ul</p></td>
| + | |
| - | <td width="0"><p align="left">200 uM</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">5X Phusion HF Buffer</p></td>
| + | |
| - | <td width="0"><p align="left">10 ul</p></td>
| + | |
| - | <td width="0"><p align="left">1X</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">ddH2O</p></td>
| + | |
| - | <td width="0"><p align="left">To 50 ul</p></td>
| + | |
| - | <td width="0"></td>
| + | |
| - | </tr>
| + | |
| - | </table></div>
| + | |
| - | <p>3. The master mix for reactions with Taq DNA polymerase contained:</p>
| + | |
| - | <div align="center"><table style="margin-left:50px" border="1" cellspacing="0" cellpadding="0" width="0">
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">Reagent</p></td>
| + | |
| - | <td width="0"><p align="left">Volume</p></td>
| + | |
| - | <td width="0"><p align="left">Final Concentration</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">10X Standard Taq Buffer</p></td>
| + | |
| - | <td width="0"><p align="left">5 ul</p></td>
| + | |
| - | <td width="0"><p align="left">1X</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">10 mM dNTPs</p></td>
| + | |
| - | <td width="0"><p align="left">1 ul</p></td>
| + | |
| - | <td width="0"><p align="left">200 uM</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">10 uM Forward Primer</p></td>
| + | |
| - | <td width="0"><p align="left">1 ul</p></td>
| + | |
| - | <td width="0"><p align="left">0.2 uM</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">10 uM Reverse Primer</p></td>
| + | |
| - | <td width="0"><p align="left">1 ul</p></td>
| + | |
| - | <td width="0"><p align="left">0.2 uM</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">Template DNA</p></td>
| + | |
| - | <td width="0"><p align="left">Variable</p></td>
| + | |
| - | <td width="0"><p align="left">Variable</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">Taq DNA polymerase</p></td>
| + | |
| - | <td width="0"><p align="left">0.25 ul</p></td>
| + | |
| - | <td width="0"><p align="left">1.25 units/50 ul PCR</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">ddH2O</p></td>
| + | |
| - | <td width="0"><p align="left">To 50 ul</p></td>
| + | |
| - | <td width="0"></td>
| + | |
| - | </tr>
| + | |
| - | </table></div>
| + | |
| - | <p>4. All temperature profiles were optimized according to manufacturer’s protocol, the melting temperature of primers, and the length of the desired PCR products.<br>
| + | |
| - | Basic temperature profiles:</p>
| + | |
| - | <div align="center"><table style="margin-left:50px" border="1" cellspacing="0" cellpadding="0" width="0">
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">Step</p></td>
| + | |
| - | <td width="0"><p align="left">Temperature</p></td>
| + | |
| - | <td width="0"><p align="left">Time</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">Initial Denaturation</p></td>
| + | |
| - | <td width="0"><p align="left">95℃ </p></td>
| + | |
| - | <td width="0"><p align="left">3 min</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0" rowspan="3"><p align="left">30 Cycles</p></td>
| + | |
| - | <td width="0"><p align="left">95℃ </p></td>
| + | |
| - | <td width="0"><p align="left">30 sec</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">55℃ </p></td>
| + | |
| - | <td width="0"><p align="left">30 sec</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">72℃ </p></td>
| + | |
| - | <td width="0"><p align="left">1 min</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">Final Extension</p></td>
| + | |
| - | <td width="0"><p align="left">72℃ </p></td>
| + | |
| - | <td width="0"><p align="left">5 min</p></td>
| + | |
| - | </tr>
| + | |
| - | <tr>
| + | |
| - | <td width="0"><p align="left">Hold</p></td>
| + | |
| - | <td width="0"><p align="left">4℃ </p></td>
| + | |
| - | <td width="0"></td>
| + | |
| - | </tr>
| + | |
| - | </table></div>
| + | |
| - | <p align="left"> </p>
| + | |
| - | <p align="left"><strong>Gel purification of DNA</strong> <strong>Fragment</strong></p>
| + | |
| - | <blockquote>
| + | |
| - | <p align="left">Agarose Electrophoresis:<br>
| + | |
| - | 1. A mixture of various sized DNA fragments were separated in an agarose gel (from 0.8 to 1.5% agarose in 1x TAE buffer ethidium bromide) at a constant voltage of 150 V.<br>
| + | |
| - | 2. UV light (λ = 254 nm) was used to visualize DNA with intercalated ethidium bromide.<br>
| + | |
| - | </p>
| + | |
| - | <p align="left">Gel Purification of DNA fragment:<br>
| + | |
| - | 1. The band with the desired DNA fragments were excised from the gel, using a clean scalpel.<br>
| + | |
| - | 2. DNA was isolated from the gel slice with Gel Extraction Kit according to the manufacturer’s protocol.<br>
| + | |
| - | 3. Purity and amount of DNA was determined using NanoDrop.</p>
| + | |
| - | </blockquote>
| + | |
| - | <p align="left"><strong>Restriction Digestion</strong></p>
| + | |
| - | <ol>
| + | |
| - | <p align="left">1. To digest the desired DNA restriction reactions were prepared as follows:<br>
| + | |
| - | For analysis of cloned DNA <br>
| + | |
| - | 2µl of the appropriate restriction buffer (10X) <br>
| + | |
| - | 0.5 µL restriction enzyme <br>
| + | |
| - | Bring volume to 20 µL with nuclease-free water. <br>
| + | |
| - | Or<br>
| + | |
| - | For isolation of specific DNA <br>
| + | |
| - | 2µl of the appropriate restriction buffer (10X) <br>
| + | |
| - | Up to 2 µL restriction enzyme <br>
| + | |
| - | Bring volume to 50 µL with nuclease-free water. <br>
| + | |
| - | 2. The sample was incubated at optimal temperature for the restriction enzymes.<br>
| + | |
| - | 3. Analysis of fragmented DNA was done by gel electrophoresis.<br>
| + | |
| - | 4. Desired DNA fragment was excised and purified using suitable DNA purification kit.</p>
| + | |
| - | <p align="left">
| + | |
| - | <section></section>
| + | |
| - | </p>
| + | |
| - | </ol>
| + | |
| - | <p align="left"><strong>Ligation</strong></p><ol>
| + | |
| - | <p>T4 ligase ligates the 5' phosphate and the 3'-hydroxyl groups of DNA.<br>
| + | |
| - | Vector and insert concentrations were estimated and insert and vector fragments joined in a molar ratio of 3:1 (100-150ng Vector DNA).<br>
| + | |
| - | A ligation mixture was prepared:<br>
| + | |
| - | 1X ligase buffer (10X) <br>
| + | |
| - | 1 µL T4 ligase (3 U/µL) <br>
| + | |
| - | Bring volume to 10 µL with nuclease-free water. <br>
| + | |
| - | Reactions were incubated at 17 °C for 4 to 18 hours. </p>
| + | |
| - | <p>After incubation part of the ligation mixture was used for the transformation of bacterial cells (see: transformation of bacteria). </p>
| + | |
| - | </ol>
| + | |
| - | <p align="left"><strong>Culturing bacteria</strong></p>
| + | |
| - | <blockquote>
| + | |
| - | <p>For plasmid DNA propagation two bacterial strains were used: DH5alpha and TransT1.</p>
| + | |
| - | <p><br>
| + | |
| - | Growth media for bacteria:<br>
| + | |
| - | Luria Broth (LB) : 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid: ampicilin 100 mg/L or kanamycin 50 mg/L.</p>
| + | |
| - | <p><br>
| + | |
| - | LB agar plates: LB with 1.5% agar, media is supplemented with suitable antibiotics depending on the selection marker on the transfected plasmid.</p>
| + | |
| - | </blockquote>
| + | |
| - | <p align="left"> </p>
| + | |
| - | <p align="left"><strong> Bacteria </strong><strong>transformation</strong></p>
| + | |
| - | <blockquote>
| + | |
| - | <p>E. coli DH5alpha and TransT1 competent cells were used for the propagation of plasmid DNA.</p>
| + | |
| - | <p> 100 µL of competent cells were thawed on ice. <br>
| + | |
| - | 50 – 400 ng DNA solution was added to competent bacterial cells (depending on the concentration of the DNA solution). <br>
| + | |
| - | A mixture of cells and DNA solution was incubated on ice for 30-60 minutes. <br>
| + | |
| - | The mixture was heat-shocked for 3 minutes at 42 °C. <br>
| + | |
| - | Cooled for 3 minutes on ice. <br>
| + | |
| - | 500 µL of preheated antibiotic free LB-medium was added and incubated for one hour at 37 °C with agitation for the purpose of inducing antibiotic resistance. <br>
| + | |
| - | The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates.
| + | |
| - | </p>
| + | |
| - | </blockquote>
| + | |
| - | <p align="left"> </p>
| + | |
| - | <p align="left"><strong>Plasmid DNA isolation</strong></p>
| + | |
| - | <blockquote>
| + | |
| - | <p>MINI PREPs for analysis and sequencing:<br>
| + | |
| - | A single colony was picked from a LB-agar plate or glycerol stock and inoculated in 10 mL of LB-medium with the appropriate antibiotic for selection (100 mg/L ampicillin, 50 mg/L kanamycin, 35 mg/L chloramphenicol). <br>
| + | |
| - | Bacteria were grown over night at 37 °C with agitation. <br>
| + | |
| - | Plasmid DNA was isolated from 6-10 mL of over-night culture with plasmid miniprep kit according to the manufacturer's protocol. <br>
| + | |
| - | Amounts ranging from 6-10 µg of plasmid DNA were obtained. <br>
| + | |
| - | The purity and concentration of the isolated DNA was analyzed using NanoDrop.</p>
| + | |
| - | </blockquote>
| + | |
| - | <p> </p>
| + | |
| - | <p style="text-align: right;">Return to: <a href="https://2014.igem.org/Team:Tsinghua/Notebook/Protocol">Notebook -> Protocol</a></p>
| + | |
| - | <p> </p>
| + | |
| - | | + | |
| | <!-- end .content --></article> | | <!-- end .content --></article> |
| | | | |
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