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| <ul class="nav"> | | <ul class="nav"> |
| <li class="active"><a href="#nav1">Restriction digestion</a></li> | | <li class="active"><a href="#nav1">Restriction digestion</a></li> |
− | <li><a href="#nav2">Ligation</a></li>
| |
− | <li><a href="#nav3">Gibson assembly</a></li>
| |
− | <li><a href="#nav4">Preparing competent <i>E. coli</i></a></li>
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− | <li><a href="#nav5">Preparing competent <i>L. lactis</i></a></li>
| |
− | <li><a href="#nav6">Transformation <i>E. coli</i></a></li>
| |
− | <li><a href="#nav7">Electrotransformation <i>L. lactis</i></a></li>
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− | <li><a href="#nav8">Colony PCR</a></li>
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− | <li><a href="#nav9">Quickchange PCR</a></li>
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− | <li><a href="#nav10">Taq PCR</a></li>
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− | <li><a href="#nav11">Phusion PCR</a></li>
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− | <li><a href="#nav12">PCR cleanup</a></li>
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− | <li><a href="#nav13">Media</a></li>
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− | <li><a href="#nav14">Antibiotic</a></li>
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| </ul> | | </ul> |
| </nav> | | </nav> |
− | <div style="margin-left:25%;">
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| <div class="main-col"> | | <div class="main-col"> |
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| <h1> Protocols</h1> | | <h1> Protocols</h1> |
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| + | <img src="https://static.igem.org/mediawiki/2017/0/03/2017-10-14_19.37.13.jpg" style="width:100%"> |
| + | <div class="slidetext"><h2 class="header_title"></h2></div> |
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| + | </div> |
| + | </div> |
| | | |
| <br> | | <br> |
− | <ol>
| |
− | <li id="nav1"><b>Restriction Digestion</b>
| |
− | <ol type=a>
| |
− | <li>Materials
| |
− | <ol type=i>
| |
− | <li>(1) 8-tube strip, or (3) 0.6ml thin-walled tubes</li>
| |
− | <li>BioBrick Part in BioBrick plasmid (Purified DNA, > 16ng/ul)</li>
| |
− | <li>dH2O</li>
| |
− | <li>NEB Buffer 2</li>
| |
− | <li>BSA</li>
| |
− | <li>Restriction Enzymes: EcoRI, SpeI, XbaI, PstI</li>
| |
− | </ol>
| |
− | <li>Procedure
| |
− | <ol type=i>
| |
− | <li>Add 250ng of DNA to be digested, and adjust with dH20 for a total volume of 16ul</li>
| |
− | <li>Add 2.5ul of NEBuffer 2.1</li>
| |
− | <li>Check here for buffer selection (depending on the enzyme)</li>
| |
− | <li>Add 0.5ul of BSA</li>
| |
− | <li>Add 0.5ul of EcoRI</li>
| |
− | <li>Add 0.5ul of PstI</li>
| |
− | <li>There should be a total volume of 20ul. Mix well and spin down briefly</li>
| |
− | <li>Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes. <i>We incubate in a thermal cycler with a heated lid</i></li>
| |
− | <li>Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.</li>
| |
− | </ol>
| |
− | </li>
| |
− | <li>Source
| |
− | <ol type=i>
| |
− | <li>iGEM http://parts.igem.org/Help:Protocols/Restriction_Digest</li>
| |
− | </ol>
| |
− | </li>
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− | </li>
| |
− | </ol>
| |
− | </li>
| |
− | <br>
| |
− | <li id="nav2"><b>Ligation</b>
| |
− | <ol type=a>
| |
− | <li>Materials
| |
− | <ol type=i>
| |
− | <li>Digested backbone & inserts</li>
| |
− | <li>T4 DNA ligase</li>
| |
− | <li>T4 DNA ligase buffer</li>
| |
− | </ol>
| |
− | </li>
| |
− | <li>Procedure
| |
− | <ol type=i>
| |
− | <li>Add 2ul of digested plasmid backbone (25 ng)</li>
| |
− | <li>Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 ul)</li>
| |
− | <li>Add equimolar amount of XbaI PstI digested fragment (< 3 ul)</li>
| |
− | <li>Molar ratios of 1:1, 1:10, 1:20 are recommended</li>
| |
− | <li>Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase</li>
| |
− | <li>Add 0.5 ul T4 DNA ligase</li>
| |
− | <li>Add water to 10 ul</li>
| |
− | <li>Ligate 16C/30 min, heat kill 80C/20 min</li>
| |
− | <li>Transform with 1-2 ul of product</li>
| |
− | </ol>
| |
− | </li>
| |
− | <li>Source
| |
− | <ol type=i>
| |
− | <li>iGEM http://parts.igem.org/Help:Protocols/Ligation</li>
| |
− | </ol>
| |
− | </li>
| |
− | </ol>
| |
− | </li>
| |
− | <br>
| |
− | <li id="nav3"><b>Gibson assembly</b>
| |
− | <ol type=a>
| |
− | <li>Materials
| |
− | <ol type=i>
| |
− | <li>Compatible Fragments</li>
| |
− | <li>Gibson Assembly Master Mix 2x</li>
| |
− | <li>Positive control (NEB)</li>
| |
− | </ol>
| |
− | </li>
| |
− | <li>Procedure
| |
− | <table style="width:100%">
| |
− | <tr>
| |
− | <th> </th>
| |
− | <th>2-3 Fragment Assembly</th>
| |
− | <th>4-6 Fragment Assembly</th>
| |
− | <th>Positive Control**</th>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Total Amount of Fragments</td>
| |
− | <td>0.02–0.5 pmols* X μl</td>
| |
− | <td>0.2–1 pmols* X μl</td>
| |
− | <td>10 μl</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Gibson Assembly Master Mix (2X)</td>
| |
− | <td>10 μl</td>
| |
− | <td>10 μl</td>
| |
− | <td>10 μl</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Deionized H2O</td>
| |
− | <td>10-X μl</td>
| |
− | <td>10-X μl</td>
| |
− | <td>0</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Total Volume</td>
| |
− | <td>20 μl***</td>
| |
− | <td>20 μl***</td>
| |
− | <td>20 μl</td>
| |
− | </tr>
| |
− | </table>
| |
− | </li>
| |
− | <li>Source
| |
− | <ol type=i>
| |
− | <li>https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale2621.pdf</li>
| |
− | </ol>
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− | </li>
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− | </ol>
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− | </li>
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− | <br>
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− | <li id="nav4"><b>Preparing competent <i>E. coli</i> DH5α cells</b>
| |
− | <ol type=a>
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− | <li>Materials
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− | <ol type=i>
| |
− | <li>Plate or stock of <i>E.coli</i> DH5α cell</li>
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− | <li>LB</li>
| |
− | <li><a class="mouseover" title="5g PEG 8000
| |
− | 1.5 mL 1M MgCl2 (or 0.30g MgCl2*6H20)
| |
− | 2.5 mL DMSO
| |
− | Add LB to 50 mL
| |
− | Filter sterilize (0.22 μm filter)">TSS buffer</a></li>
| |
− | <li>Ice</li>
| |
− | </ol>
| |
− | </li>
| |
− | <li>Procedure
| |
− | <ol type=i>
| |
− | <li>Grow 5ml overnight culture of cells in LB media, dilute this culture back into 25-50ml of fresh LB media in a 200ml conical flask.</li>
| |
− | <li>In the morning You should aim to dilute the overnight culture by at least 1/100.</li>
| |
− | <li>Grow the diluted culture to an OD600 of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD600 0.2)</li>
| |
− | <li>Put Eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is X ml, you will need X tubes. At this point you should also make sure that your TSS is being cooled (it should be stored at 4°C but if you have just made it fresh then put it in an ice bath).</li>
| |
− | <li>Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.</li>
| |
− | <li>All subsequent steps should be carried out at 4°C and the cells should be kept on ice whenever possible.</li>
| |
− | <li>Centrifuge for 10 minutes at 3000 rpm and 4°C.</li>
| |
− | <li>Decant supernatant, remove leftover media by carefully pipetting</li>
| |
− | <li>Resuspend in TSS buffer (10% of original volume), vortex gently</li>
| |
− | <li>Add 100 μl aliquots to chilled Eppendorfs, flash freeze and store at – 80°C in 200 µl aliquots.</li>
| |
− | </ol>
| |
− | </li>
| |
− | <li>Source
| |
− | <ol type=i>
| |
− | <li>https://openwetware.org/wiki/Preparing_chemically_competent_cells</li>
| |
− | </ol>
| |
− | </li>
| |
− | </ol>
| |
− | </li>
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− | <br>
| |
− | <li id="nav5"><b>Making competent Lactococcus <i>L. lactis</i> NZ9000 cells</b>
| |
− | <ol type=a>
| |
− | <li>Materials
| |
− | <ol type=i>
| |
− | <li><a class="mouseover" title="M17
| |
− | 0.5% glucose">GM17</a></li>
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− | <li><a class="mouseover" title="M17
| |
− | 0.5 M sucrose
| |
− | 0.5 % glucose
| |
− | 2% glycine">SGM17 + glycine</a></li>
| |
− | <li><a class="mouseover" title="0.5 M sucrose
| |
− | 10% glycerol">Electroporation buffer</a></li>
| |
− | </ol>
| |
− | </li>
| |
− | <li>Procedure
| |
− | <ol type=a>
| |
− | <li>Grow cells overnight in 25ml of GM17</li>
| |
− | <li>Add 1ml of overnight culture into 25ml SGM17 + glycine</li>
| |
− | <li>Grow for ~4 hours until OD600 ~ 0.7</li>
| |
− | <li>Chill culture on ice for 10 mins</li>
| |
− | <li>Centrifuge cells for 15 mins at 3000g</li>
| |
− | <li>Gently shake to resuspend pellet in 3ml Electroporation Buffer</li>
| |
− | <li>Centrifuge cells for 15 mins at 3000g</li>
| |
− | <li>Resuspend pellet in 3ml Electroporation Buffer</li>
| |
− | <li>Centrifuge cells for 15 mins at 3000g</li>
| |
− | <li>Resuspend pellet in 500 µl Electroporation Buffer</li>
| |
− | <li>Separate into 100 µl aliquots and store at -80°C until use.</li>
| |
− | </ol>
| |
− | </li>
| |
− | <li>Source
| |
− | <ol type=i>
| |
− | <li>https://openwetware.org/wiki/Lactococcus_transformation</li>
| |
− | </ol>
| |
− | </li>
| |
− | </ol>
| |
− | <br>
| |
− | </li>
| |
− | <li id="nav6"><b>Transformation <i>E.coli</i> DH5α</b>
| |
− | <ol type=a>
| |
− | <li>Materials
| |
− | <ol type=i>
| |
− | <li>Plasmid DNA</li>
| |
− | <li>Competent <i>E. coli</i> DH5α cells: 50 µl per transformation</li>
| |
− | <li><a class="mouseover" title="1% Yeast Extract
| |
− | 4% Tryptone
| |
− | 40 mM Glucose
| |
− | Store at 4°C">2x SOC stock</a></li>
| |
− | <li><a class="mouseover" title="1 M NaCl
| |
− | 250 mM KCl
| |
− | 1 M MgCl2
| |
− | 1 M MgSO4
| |
− | Store at 4°C">SOC salt stocks</a></li>
| |
− | <li>Sterile MQ</li>
| |
− | <li>LB agar selection plates: Two per transformation</li>
| |
− | <li>Eppendorf tubes</li>
| |
− | <li>Floater</li>
| |
− | <li>Ice</li>
| |
− | <li>42°C water bath</li>
| |
− | <li>37°C incubator: both shaker and stove</li>
| |
− | <li>Sterile spreader/glass beads</li>
| |
− | </ol>
| |
− | </li>
| |
− | <li>Procedure (on ice)
| |
− | <ol type=i>
| |
− | <li>Thaw competent cells on ice. When using multiple aliquots, first pool all cells into a single volume to homonogize the solution. Dispose of unused competent cells. Do not refreeze since reusing thawed cells, will drastically reduce transformation efficiency.</li>
| |
− | <li>Pipet 50 µl of competent cells into Eppendorf tube for each transformation (labeled, prechilled, in floating rack), don’t forget control tubes</li>
| |
− | <li>Pipet 1-100 ng of DNA as well as control into tubes and gently mix with tip</li>
| |
− | <li>incubate on ice for 30 min, tubes may be gently flicked, return to ice ASAP</li>
| |
− | <li>Meanwhile, for every transformation, add 2 µl of each SOC salt solution into 100 µl 2x SOC stock and ad to 200 µl with sterile MQ. Place the SOC medium on ice till use</li>
| |
− | <li>Heat shock tubes at 42°C for 30 seconds (precisely)</li>
| |
− | <li>Incubate on ice for 5 min</li>
| |
− | <li>Add 200 µl of SOC medium to each transformation</li>
| |
− | <li>Incubate at 37°C for 1 hours, shaker or rotor recommended</li>
| |
− | <li>Pipet 20 µl & 200 µl transformation mixture onto petri plates and spread with sterilized spreader or glass beads. Let the plates dry near the flame before placing them in the incubator</li>
| |
− | <li>Incubate plates upside down overnight (14-18hr) at 37°C</li>
| |
− | <li>Pick single colonies</li>
| |
− | <li>Perform Colony PCR to verify</li>
| |
− | <li>Grow cells & miniprep</li>
| |
− | <li>Calculate efficiency by counting colonies (expected value: 1.5x10^8 to 6x10^8 cfu/µg DNA)</li>
| |
− | </ol>
| |
− | </li>
| |
− | <li>Source
| |
− | <ol type=i>
| |
− | <li>http://parts.igem.org/Help:Protocols/Transformation</li>
| |
− | </ol>
| |
− | </li>
| |
− | </ol>
| |
− | </li>
| |
− | <br>
| |
− | <li id="nav7"><b>Electrotransformation <i>L. Lactis</i></b>
| |
− | <ol type=a>
| |
− | <li>Materials
| |
− | <ol type=i>
| |
− | <li>Plasmid DNA (preferably without salts from buffers. These can be removed by incubating the DNA on a filter, which is floating on MQ for 10 minutes at room temperature</li>
| |
− | <li>Electrocompetent <i>L. lactis</i> cells: 50 µl per transformation</li>
| |
− | <li><a class="mouseover" title="M17
| |
− | 0.5% glucose
| |
− | 0.5 M sucrose
| |
− | 2 mM MgCl2
| |
− | 2 mM CaCl2">Recovery medium</a></li>
| |
− | <li>Electroporation Cuvettes</li>
| |
− | <li>Electroporator</li>
| |
− | <li>SGM17 agar selection plates</li>
| |
− | <li>Eppendorf tubes</li>
| |
− | <li>Ice</li>
| |
− | <li>Eppendorf centrifuge</li>
| |
− | <li>30°C incubator</li>
| |
− | <li>Sterile spreader/glass beads</li>
| |
− | </ol>
| |
− | </li>
| |
− | <li>Procedure
| |
− | <ol type=i>
| |
− | <li>Mix all elements of the recovery medium (1 ml per transformation) and place it on ice together with the electroporation cuvettes</li>
| |
− | <li>Thaw competent cells on ice. When using multiple aliquots, first pool all cells into a single volume to homonogize the solution. Dispose of unused competent cells. Do not refreeze since reusing thawed cells, will drastically reduce transformation efficiency.</li>
| |
− | <li>Pipet 50 µl of competent cells into Eppendorf tube for each transformation (labeled, prechilled, in floating rack), don’t forget control tubes</li>
| |
− | <li>Add at most 5 µl of plasmid DNA to the competent cells and incubate on ice for 10 minutes</li>
| |
− | <li>Dry the cuvette, electroporate at 2500 V and carefully add 950 µl recovery medium to the cells. Place the cuvette back on ice and incubate for 10 minutes</li>
| |
− | <li>Transfer the cell suspension with careful mixing into Eppendorf tubes and incubate for 2 hours at 30°C</li>
| |
− | <li>Plate 100 µl of the recovered cells onto a selection plate and spin down the remaining cells at 6000 rpm for 5 minutes</li>
| |
− | <li>Resuspend cell pellet into 50-100µl and plate on a selection plate</li>
| |
− | <li>Incubate the plates at 30°C for 24 - 48 hours</li>
| |
− | </ol>
| |
− | </li>
| |
− | <li>Source
| |
− | <ol type=i>
| |
− | <li>https://openwetware.org/wiki/Lactococcus_transformation</li>
| |
− | </ol>
| |
− | </li>
| |
− | </ol>
| |
− | </li>
| |
− | <br>
| |
− | <li id="nav8"><b>Colony PCR</b>
| |
− | <ol type=a>
| |
− | <li>Materials
| |
− | <ol type=i>
| |
− | <li>10X Standard Taq Reaction Buffer</li>
| |
− | <li>10 mM dNTPs</li>
| |
− | <li>10 µM Forward Primer</li>
| |
− | <li>10 µM Reverse Primer</li>
| |
− | <li>Template DNA (colony resuspended in MQ / plasmid DNA)</li>
| |
− | <li>Taq DNA Polymerase</li>
| |
− | <li>Nuclease-free water</li>
| |
− | <li>PCR tubes</li>
| |
− | <li>Ice</li>
| |
− | <li>PCR tube rack</li>
| |
− | </ol>
| |
− | </li>
| |
− | <li>Procedure
| |
− | <ol type=i>
| |
− | <li>Suspend a colony in 10 µl sterile MQ</li>
| |
− | <li>Prepare Mastermix for 10 reactions according to:
| |
− | <table style="width:100%">
| |
− | <tr>
| |
− | <th>Component</th>
| |
− | <th>220 µl = 10 colonies</th>
| |
− | <th>Final Concentration</th>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>10X Standard Taq (Mg-free) Reaction Buffer</td>
| |
− | <td>22 µl</td>
| |
− | <td>1X</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>25 mM MgCl2</td>
| |
− | <td>13,2 µl</td>
| |
− | <td>1.5 mM</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>10 mM dNTPs</td>
| |
− | <td>4,4 µl</td>
| |
− | <td>200 µM</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>10 µM pJET fw</td>
| |
− | <td>4,4 µl</td>
| |
− | <td>0.2 µM (0.05–1 µM, typically 0.1-0.5µM)</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>10 µM pJET rv</td>
| |
− | <td>4,4 µl</td>
| |
− | <td>0.2 µM (0.05–1 µM, typically 0.1-0.5µM)</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Taq DNA Polymerase</td>
| |
− | <td>1,1 µl</td>
| |
− | <td>1.25 units/50 µl PCR</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Nuclease-free water</td>
| |
− | <td>148,5 µl</td>
| |
− | <td>-</td>
| |
− | </tr>
| |
− | </table>
| |
− | </li>
| |
− | <li>Put 19 µl of mastermix in each reaction tube & add 2 µl suspended colony mixture</li>
| |
− | <li>Add 2 µl plasmid DNA for positive control and 2 µl MQ for the negative control</li>
| |
− | <li>Place the tubes in the PCR machine (Taq program)</li>
| |
− | <li>Once the PCR is done, mix 10 µl of PCR product with 2 µl 6X purple gel loading dye and run it on a gel for 50 minutes at 130 Volts</li>
| |
− | <li>If the correct products are present in the gel samples, inoculate overnight cultures from the original plates.</li>
| |
− | <li>Mix 5 ml LB with appropriate antibiotic. Scoop a colony from the plate and drop the tip into the medium. Incubate the tube at 37°C overnight to let the culture grow</li>
| |
− | </ol>
| |
− | </li>
| |
− | <li>Source
| |
− | <ol type=i>
| |
− | <li>https://www.qiagen.com/us/resources/download.aspx?id=c73208eb-a83e-40c4-a9b6-ea5c4c94b9f4&lang=en</li>
| |
− | </ol>
| |
− | </li>
| |
− | </ol>
| |
− | </li>
| |
− | <br>
| |
− | <li id="nav9"><b>Quickchange PCR</li></b>
| |
− | <br>
| |
− | <li id="nav10"><b>Taq PCR</b>
| |
− | <ol type=a >
| |
− | <li>Materials according to table</li>
| |
− | <li>Procedure
| |
− | <ol type=i>
| |
− | <li>Mix according to table
| |
− | <table style="width:100%">
| |
− | <tr>
| |
− | <th>Component</th>
| |
− | <th>25 μl reaction</th>
| |
− | <th>50 μl reaction</th>
| |
− | <th>Final Concentration</th>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>10X Standard Taq Reaction Buffer</td>
| |
− | <td>2.5 μl</td>
| |
− | <td>5 μ</td>
| |
− | <td>1X</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>10 mM dNTPs</td>
| |
− | <td>0.5 µl</td>
| |
− | <td>1 μl</td>
| |
− | <td>200 µM</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>10 µM Forward Primer</td>
| |
− | <td>0.5 µl</td>
| |
− | <td>1 μl</td>
| |
− | <td>0.2 µM (0.05–1 µM)</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>10 µM Reverse Primer</td>
| |
− | <td>0.5 µl</td>
| |
− | <td>1 μl</td>
| |
− | <td>0.2 µM (0.05–1 µM)</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Template DNA</td>
| |
− | <td>variable</td>
| |
− | <td>variable</td>
| |
− | <td>1,000 ng</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Taq DNA Polymerase</td>
| |
− | <td>0.125 µl</td>
| |
− | <td>0.25 µl</td>
| |
− | <td>1.25 units/50 µl PCR</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Nuclease-free water</td>
| |
− | <td>to 25 µl</td>
| |
− | <td>to 50 µl</td>
| |
− | <td>-</td>
| |
− | </tr>
| |
− | </table>
| |
− | </li>
| |
− | <li>PCR cycler conditions
| |
− | <table style="width:100%">
| |
− | <tr>
| |
− | <th>Step</th>
| |
− | <th>Temperature</th>
| |
− | <th>Time</th>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Initial Denaturation</td>
| |
− | <td>95°C</td>
| |
− | <td>30 sec</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>30 cycles</td>
| |
− | <td>95°C</td>
| |
− | <td>15-30 sec</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>30 cycles</td>
| |
− | <td>45°C-68°C</td>
| |
− | <td>15-60 sec</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>30 cycles</td>
| |
− | <td>68°C</td>
| |
− | <td>1 min/ kb</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Final extension</td>
| |
− | <td>68°C</td>
| |
− | <td>5 min</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Hold</td>
| |
− | <td>4-10°C</td>
| |
− | <td>-</td>
| |
− | </tr>
| |
− | </table>
| |
− | </li>
| |
− | </ol>
| |
− | <li>Source
| |
− | <ol type=i>
| |
− | <li>NEB https://www.neb.com/protocols/1/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273</li>
| |
− | </ol>
| |
− | </li>
| |
− | </li>
| |
− | </ol>
| |
− | </li>
| |
− | <br>
| |
− | <li id="nav11"><b>Phusion PCR</b>
| |
− | <ol type=a >
| |
− | <li>Materials according to table</li>
| |
− | <li>Procedure
| |
− | <table style="width:100%">
| |
− | <tr>
| |
− | <th>Component</th>
| |
− | <th>20 μl reaction</th>
| |
− | <th>50 μl reaction</th>
| |
− | <th>Final Concentration</th>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>5X Phusion HF/ GC Buffer</td>
| |
− | <td>4 μl</td>
| |
− | <td>10 μl</td>
| |
− | <td>1X</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>10 mM dNTPs</td>
| |
− | <td>0.4 µl</td>
| |
− | <td>1 μl</td>
| |
− | <td>200 µM</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>10 µM Forward Primer</td>
| |
− | <td>1 µl</td>
| |
− | <td>2,5 μl</td>
| |
− | <td>0.5 µM (0.05–1 µM)</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>10 µM Reverse Primer</td>
| |
− | <td>1 µl</td>
| |
− | <td>2,5 μl</td>
| |
− | <td>0.5 µM (0.05–1 µM)</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Template DNA</td>
| |
− | <td>variable</td>
| |
− | <td>variable</td>
| |
− | <td><250 ng</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Phusion DNA Polymerase</td>
| |
− | <td>0.2 µl</td>
| |
− | <td>0.5 µl</td>
| |
− | <td>1. units/50 µl PCR</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Nuclease-free water</td>
| |
− | <td>to 20 µl</td>
| |
− | <td>to 50 µl</td>
| |
− | <td>-</td>
| |
− | </tr>
| |
− | </table>
| |
− | </li>
| |
− | <li>PCR cycler conditions</li>
| |
− | <li>
| |
− | <table style="width:100%">
| |
− | <tr>
| |
− | <th>Step</th>
| |
− | <th>Temperature</th>
| |
− | <th>Time</th>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Initial Denaturation</td>
| |
− | <td>98°C</td>
| |
− | <td>30 sec</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>30 cycles</td>
| |
− | <td>98°C</td>
| |
− | <td>5-10 sec</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>30 cycles</td>
| |
− | <td>45°C-72°C</td>
| |
− | <td>10-30 sec</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>30 cycles</td>
| |
− | <td>72°C</td>
| |
− | <td>15-30 sec/ kb</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Final extension</td>
| |
− | <td>72°C</td>
| |
− | <td>5-10 min</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Hold</td>
| |
− | <td>4-10°C</td>
| |
− | <td>-</td>
| |
− | </tr>
| |
− | </table>
| |
− | </li>
| |
− | <li>Source
| |
− | <ol type=i>
| |
− | <li>https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530</li>
| |
− | </ol>
| |
− | </li>
| |
− | </ol>
| |
− | </li>
| |
− | <br>
| |
− | <li id="nav12"><b>PCR cleanup, Gel extraction, minipreps was performed by using kits provided by Qiagen. Standard procedure was followed.</b></li>
| |
− | <br>
| |
− | <li id="nav13"><b>Media</b>
| |
− | <ol type=a>
| |
− | <li>Lysogeny Broth (LB)
| |
− | <ol type=i>
| |
− | <li>Dissolve the following in 1L ddH2O</li>
| |
− | <li>10g bacto-tryptone</li>
| |
− | <li>5g bacto-yeast extract</li>
| |
− | <li>10g NaCl (5g in some recipes)</li>
| |
− | <li>Adjust to pH 7.0 with NaOH</li>
| |
− | </ol>
| |
− | </li>
| |
− | <li>M17
| |
− | <ol type=i>
| |
− | <li>Dissolve the following in 1L ddH2O</li>
| |
− | <li>5.0 g Pancreatic Digest of Casein</li>
| |
− | <li>5.0 g Soy Peptone</li>
| |
− | <li>5.0 g Beef Extract</li>
| |
− | <li>2.5 g Yeast Extract</li>
| |
− | <li>0.5 g Ascorbic Acid</li>
| |
− | <li>0.25 g Magnesium Sulfate</li>
| |
− | <li>10.0 g Disodium-β-glycerophosphate</li>
| |
− | <li>11.0 g Agar</li>
| |
− | </ol>
| |
− | </li>
| |
− | <li>SOC
| |
− | <ol type=i>
| |
− | <li>Dissolve the following in 1L ddH2O</li>
| |
− | <li>20g Bacto Tryptone</li>
| |
− | <li>5g Bacto Yeast Extract</li>
| |
− | <li>2ml of 5M NaCl</li>
| |
− | <li>2.5ml of 1M KC</li>
| |
− | <li>10ml of 1M MgCl2</li>
| |
− | <li>10ml of 1M MgSO4</li>
| |
− | <li>20ml of 1M glucose</li>
| |
− | </ol>
| |
− | </li>
| |
− | </ol>
| |
− | </li>
| |
− | <br>
| |
− | <li id="nav14"><b>Antibiotic</b>
| |
− | <ol type=a>
| |
− | <li>Chloramphenicol</li>
| |
− | <li>Erythromycin</li>
| |
− | <li>Ampicillin</li>
| |
− | </ol>
| |
− | </li>
| |
− | </ol>
| |
| | | |
− | </div> <!-- margin --> | + | <div style="text-align:center"> |
| + | <span class="dot"></span> |
| + | <span class="dot"></span> |
| + | <span class="dot"></span> |
| + | <span class="dot"></span> |
| + | <span class="dot"></span> |
| + | <span class="dot"></span> |
| + | <span class="dot"></span> |
| + | <span class="dot"></span> |
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| </div> <!-- main-col --> | | </div> <!-- main-col --> |
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