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| | <h1>Basic Molecular Biology Techniques:</h1> | | <h1>Basic Molecular Biology Techniques:</h1> |
| − | <h3>PCR using Q5 Hi-Fi DNA polymerase Master Mix</h3> | + | <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_PCR"><h3>PCR using Q5 Hi-Fi DNA polymerase Master Mix</h3></a> |
| − | <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2017/6/62/XJTLU_primer.png" width="500" heighth="500" > | + | <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_PCR_purification"><h3>PCR purification using QIAquick PCR purification kit</h3></a> |
| − | <p>Reaction setup:</p>
| + | <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Gel_extraction"><h3>Gel Extraction</h3></a> |
| − | <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2017/d/d9/Reaction_setup.png" width="600" heighth="500"> | + | <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Gibson_Assembly"><h3>Gibson assembly</h3></a> |
| − | <p>The template DNA should keep an amount of 1-1000 ng</p>
| + | <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Digestion"><h3>Digestion (NEB enzymes)</h3></a> |
| − | <p>Thermocycler conditions</p>
| + | <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Ligation"><h3>Ligation</h3></a> |
| − | <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2017/1/13/Thermocycler_conditions.png" width="500" heighth="500"> | + | <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Transformation"><h3>Transformation of plasmids in <i>E.coli</i> DH5ɑ</h3></a> |
| − | <p>Clarification:</p>
| + | <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Isolation"><h3>Isolation of plasmids in <i>E.coli</i> DH5ɑ</h3></a> |
| − | <ol>
| + | <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Preparation_Competent"><h3>Preparation of competent <i>Lactococcus lactis</i></h3></a> |
| − | <li>The sequences in red are where the primers anneal with the biobrick affixes. And the 5’overhangs side with the sequences in red are designed for Gibson Assembly with linearized pNZ8148 digested by PstI due to a 21-nucleotide overlap between them.</li> | + | <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Electroporation"><h3>Electroporation for <i>Lactococcus lactis</i></h3></a> |
| − | <li>The reason of setting two different cycles is that, at the beginning of PCR, the annealed parts between primers and templates are only sequences in red. As PCR proceeds, 5’overhangs are added to the entire genes, so the rest reaction of PCR becomes the one between the whole primers and the whole genes, because of which the Tm varies compared to the former.</li>
| + | <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Plasmid_DNA_isolation"><h3>Plasmid DNA isolation from <i>Lactococcus lactis</i></h3></a> |
| − | </ol>
| + | <a href="https://2017. igem. org/ Team:XJTLU-CHINA/Protocols_Dot_blot">< h3> Dot Blot</ h3></ a> |
| − | <p>Applications in this project: Amplification of BBa_K2309028, BBa_K2309003 and BBa_K2309004</p> | + | <hr> |
| − | <hr>
| + | <h1>Anti-microbial peptides assay:</h1> |
| − | <h3>PCR purification using QIAquick PCR purification kit</h3>
| + | <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_CFU_measurement"><h3>Pre-experiment: CFU measurement</h3></a> |
| − | <ol>
| + | <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Inhibition_ring"><h3>Inhibition disc</h3></a> |
| − | <li>Add 500 μl Qiagen buffer PB</li>
| + | |
| − | <li>Spin through a column twice, discard flow-through</li> | + | |
| − | <li>Wash 1x with 700 μl buffer PB</li>
| + | |
| − | <li>Wash 2x with 760 μl buffer PE</li>
| + | |
| − | <li>Discard liquid, spin dry at 17000g for 3 min</li>
| + | |
| − | <li>Elute into a new tube twice with 50 μl of TE (100 μl total)</li> | + | |
| − | </ol>
| + | |
| − | <p>(Can be also referred to from iGEM protocol)</p>
| + | |
| − | <hr>
| + | |
| − | <h3>Gel extraction</h3>
| + | |
| − | <p>Reagents:</p> | + | |
| − | <p>1.5 ml micro-centrifuge tube; 2 ml collection tube; a QIAquick spin column; Isopropanol (100%); Buffer QG; Buffer PE; Buffer EB
| + | |
| − | </p>
| + | |
| − | <p>Procedure:</p>
| + | |
| − | <ol>
| + | |
| − | <li>Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.</li>
| + | |
| − | <li>Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel ~ 100 μl). The maximum amount of gel per spin column is 400 mg. For >2% agarose gels, add 6 volumes Buffer QG.</li> | + | |
| − | <li>Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.</li>
| + | |
| − | <li>Add 1 gel volume isopropanol to the sample and mix.</li>
| + | |
| − | <li>Place a QIAquick spin column in a provided 2 ml collection tube or into a vacuum manifold. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold until all the samples have passed through the column. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes of >800 μl, load and spin/apply vacuum again.</li> | + | |
| − | <li>If DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 500 μl Buffer QG to the QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.</li>
| + | |
| − | <li>To wash, add 750 μl Buffer PE to QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow through and place the QIAquick column back into the same tube.</li>
| + | |
| − | <p>Note: If the DNA will be used for salt-sensitive applications (e.g., sequencing, blunt- ended ligation), let the column stand 2–5 min after addition of Buffer PE. Centrifuge the QIAquick column in the provided 2 ml collection tube for 1 min to remove residual wash buffer.</p>
| + | |
| − | <li>Place QIAquick column into a clean 1.5 ml microcentrifuge tube.</li>
| + | |
| − | <li>To elute DNA, add 50 μl Buffer EB (10 mM Tris•Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 μl Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time to up to 4 min can increase the yield of purified DNA.</li>
| + | |
| − | </ol>
| + | |
| − | <hr>
| + | |
| − | <h3>Gibson Assembly</h3> | + | |
| − | <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/3/32/Assembly_Protocol.png"> | + | |
| − | <p>Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases）</p>
| + | |
| − | <p>Applications in our project: Assembly of BBa_K2309004, BBa_K2309005, BBa_K2309022 and BBa_K2309028 with pNZ8148 respectively.</p>
| + | |
| − | <p>Procedure:</p> | + | |
| − | <ol>
| + | |
| − | <li>Prepare an ice box, put every material on ice.</li>
| + | |
| − | <li>Add 0.02-0.5 pmols DNA fragment (which has been amplified by PCR using primer 1 and primer 2—see at PCR protocol) into PCR tube. (The mass of DNA can be calculated by NEB-calculator)</li>
| + | |
| − | <li>Add 1/3 amount of DNA inserts PstI-digested pNZ8148 plasmid in the tube.</li>
| + | |
| − | <li>Add 2X Gibson Assembly Master Mix (produced by NEB) to the tube.</li>
| + | |
| − | <li>Fill the tube with dH<sub>2</sun>O to a total volume of 20 μl.</li>
| + | |
| − | <li>Incubate the samples in a thermocycler at 50℃ for 15 minutes. (can be longer) Store samples on ice or at -20℃ for subsequent examination or Transformation.</li>
| + | |
| | | | |
| − | </ol> | + | <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_AMPs_efficiency_MIC_assay"><h3>AMPs minimal bactericidal concentration (MBC) assay</h3></a> |
| | + | <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Biofilm_formation"><h3>Biofilm formation test</h3></a> |
| | + | <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Growth_curve"><h3>Growth curve measurement</h3></a> |
| | <hr> | | <hr> |
| − | <h3>Digestion (NEB enzymes)</h3> | + | <h1>Nisin induction of gene expression in <i>Lactococcus lactis</i>:</h1> |
| − | <ul>
| + | <a href="https://2017.igem.org/Team:XJTLU-CHINA/Protocols_Testing_sensing_pathway"><h3>Testing of <i>S. aureus</i> sensing pathway</h3></a> |
| − | <li>Single-enzyme digestion</li>
| + | <a href="https:// 2017.igem.org/ Team: XJTLU- CHINA/ Protocols_Autolysis_test"><h3> Autolysis test</h3></ a> |
| − | <p>Reagents:</p>
| + | |
| − | <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2017/f/f6/Single-enzyme_reagents.png" width="500" height="500"> | + | |
| − | <p>Usually the volume of reaction can vary:</p>
| + | |
| − | <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2017/5/5e/Vary.png" width="500" height="500">
| + | |
| − | <p>Procedure:</p>
| + | |
| − | <ol>
| + | |
| − | <li>Add 100-1000 ng DNA into an empty 1.5 ml microcentrifuge tube. </li>
| + | |
| − | <li>Add 5μl 10×NEB buffer (dependent on enzymes) to the tube.</li>
| + | |
| − | <li>Complement ddH<sub>2</sub>O to a total volume of 49 μl system.</li>
| + | |
| − | <li>At last, add 1μl restriction enzyme and incubate the reaction system at proper temperature (normally 37 ℃) for 1 hour. The digestion efficiency can be increased through a prolonged incubation time.</li>
| + | |
| − | </ol>
| + | |
| − | <p>(The work conditions of corresponding restriction enzyme can be referenced on NEB official website)</p>
| + | |
| − | <p>Application in this project: pNZ8148 plasmid digestion by PstI</p>
| + | |
| − | <li>Double-enzyme digest</li>
| + | |
| − | </ul>
| + | |
| − | <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2017/1/18/Double_digestion.png" width="500" height="500">
| + | |
| − | <p>Incubate at 37℃ for 1 hour. Heat kill at 80℃ for 20 minutes.</p>
| + | |
| − | <p>(0.5 μl BSA can be added in reaction to increase digest efficiency, and buffer should be decided depending on the enzymes used to maximize the cutting efficiency)</p>
| + | |
| − | <p>Applications in this project: PstI and EcoRI for Linearized pSB1C3, PstI and EcoRI for BBa_2309022, BBa_2309023 and BBa_2309027.</p>
| + | |
| − | <hr>
| + | |
| − | <h3>Ligation</h3>
| + | |
| − | <p>Procedure:</p>
| + | |
| − | <ol>
| + | |
| − | <li>Add 2μl digested plasmid backbone (25ng).</li>
| + | |
| − | <li>Add equimolar digested fragment.</li>
| + | |
| − | <li>Add 1μl T4 DNA ligase buffer.</li>
| + | |
| − | <li>Add 0.5μl T4 DNA ligase.</li>
| + | |
| − | <li>Add ddH<sub>2</sub>O to 10ul volume.</li>
| + | |
| − | <li>Ligate at 16℃ for 30 minutes. Heat kill at 80℃ for 20 minutes.</li>
| + | |
| − | <li>Transform 1-2μl of the product.</li>
| + | |
| − | | + | |
| − | </ol>
| + | |
| − | <hr>
| + | |
| − | <h3>Transformation of plasmids in <i>E.coli</i> DH5ɑ</h3>
| + | |
| − | <p>(Details can be referenced at iGEM official protocol)</p>
| + | |
| − | <p>Applications in this project: Cloning of BBa_K515005 </p>
| + | |
| − | <h3>Isolation of plasmid in E.coli DH5ɑ</h3>
| + | |
| − | <h3>Preparation of competent Lactococcus lactis</h3>
| + | |
| − | <h3>Electroporation for L. lactis</h3>
| + | |
| − | <h3>Plasmid DNA isolation from Lactococcus lactis</h3>
| + | |
| − | <h1>Anti-microbial peptides assay:</h1>
| + | |
| − | <h1>Nisin induction of gene expression in Lactococcus lactis:</h1>
| + | |
| − |
| + | |
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