|
|
| Line 23: |
Line 23: |
| | <div class="text_block text"> | | <div class="text_block text"> |
| | <h1> | | <h1> |
| − | Protocols
| + | |
| − | </h1> | + | |
| − | <div id="protocols-bio">
| + | |
| − | <h2>
| + | |
| − | Biolab Protocols
| + | |
| − | </h2>
| + | |
| − | <div class="protocols-list active">
| + | |
| − | <div id="agarose-gel" class="protocol">
| + | |
| − | <h3>Agarose gel</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">AG Ignatova</p>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | 0.5 g Agarose
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | 50 mL TAE-buffer (gel room)
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <ol>
| + | |
| − | <li>
| + | |
| − | Heat in microwave until agarose is dissolved
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Transfer into gel slide, tip pipet into EtBr, then into gel
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Cool for 30 minutes
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div id="backbone dephosphorylation" class="protocol">
| + | |
| − | <h3>Backbone dephosphorylation</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">AG Ignatova</p>
| + | |
| − | <p>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 10 μL
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | pSB1C3 (cut)
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 1 μL
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | FastAP
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 4 μL
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | FastAP Buffer
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </p>
| + | |
| − | <p>
| + | |
| − | → 37 °C for 30 minutes
| + | |
| − | </p>
| + | |
| − | <p>
| + | |
| − | → purify with PCR clean up kit
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div id="chloramphenicol-stock" class="protocol">
| + | |
| − | <h3>Chloramphenicol Stock</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">AG Ignatova</p>
| + | |
| − | <p>
| + | |
| − | 34 mg/mL preparation of Choramphenicol Stock (3 in EtOH)
| + | |
| − | <ol>
| + | |
| − | <li>
| + | |
| − | dissolve 34 mg of Chloramphenicol in 1 mL 100% ethanol
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | filter through a 0.22 μL filter to sterilize
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | use at 1:1000 dilution in LB or LB-agar
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | mark as CAmp (top) and CAmp/dd.mm.yy/34 mg/mL (side) in Antibiotics
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | store at -20 °C
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div id="competent-cells" class="protocol">
| + | |
| − | <h3>Competent Cells</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">Zhang Gong</p>
| + | |
| − | Buffers and solutions:
| + | |
| − | <ul class="buffers">
| + | |
| − | <li>
| + | |
| − | <p><b>Ca/glycerol buffer:</b> 60mM CaCl<sub>2</sub>, 10mM PIPES, 150mL glycerol, Fill water up to 1L, pH=7.0 <br />Filter sterilization. Avoid autoclaving.</p>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | Protocol:
| + | |
| − | <ol>
| + | |
| − | <li>
| + | |
| − | Grow the Cells at 37°C till OD=0.2-0.4
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Chill the culture on ice for 5min.
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Cllect cells by centrifugation, 6000rpm 10min 4°C
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Gently resuspend the cells from 500ml LB in 40ml cold Ca/glycerol buffer on ice.
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Incubate the cells on ice for 5~30min.
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Repeat step 3 and 4.
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Incubate the resuspended cells in Ca/glycerol buffer on ice for 30min. (The longer, the better)
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Collect cells by centrifugation, 6000rpm 10min 4°C
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Resuspend cells in 6ml Ca/glycerol buffer.
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Aliquote 150µl/tube. Freeze in liquid N<sub>2</sub> and store at -80°C.
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div id="GeneJET-PCR-Purification-Kit" class="protocol">
| + | |
| − | <h3>GeneJET PCR Purification Kit</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <h5>#K0701, #K0702</h5>
| + | |
| − | <p class="author">Thermo SCIENTIFIC Product Information</p>
| + | |
| − | <p><h3>PURIFICATION PROTOCOLS</h3></p>
| + | |
| − | <p>
| + | |
| − | <b>
| + | |
| − | Note
| + | |
| − | </b>
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | Read IMPORTANT NOTES on p.3 before starting.
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | All purification steps ahould be carried out at <b>room temperature.</b>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | All centrifugations should be carried out in a table-top microcentrifuge at <b>>12000 x g</b> (10000-14000 rpm, depending on the rotor type).
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <p>
| + | |
| − | <b>
| + | |
| − | Protocol A. DNA purification using centrifuge
| + | |
| − | </b>
| + | |
| − | </p>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <th>
| + | |
| − | Step
| + | |
| − | </th>
| + | |
| − | <th>
| + | |
| − | Procedure
| + | |
| − | </th>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 1
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | Add a <b>1:1 volume</b> of <b>Binding Buffer</b> to completed PCR mixture (e.g. for every 100 µL of Binding Buffer). Mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 2
| + | |
| − | <br /> for DNA ≥500 bp
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <i>Optional:</i> if the DNA fragment is ≥ 500 bp, add a 1:2 volume of 100% isopropanol (e.g., 100 µL of isopropanol should be added to 100 µL of PCR mixture combined with 100 µL of Binding Buffer). Mix thoroughly.
| + | |
| − | <br />
| + | |
| − | <small>
| + | |
| − | <b>Note.</b> If PCR mixture contains primer-dimers, purification without isopropanol is recommended. However, the yield of the target DNA fragment will be lower.
| + | |
| − | </small>
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 3
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | Transfer up to 800 µL of the solution from step 1 (or optional step 2) to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through.
| + | |
| − | <br />
| + | |
| − | <small>
| + | |
| − | <b>Note.</b> If the total volume exceeds 800 µL, the solution can be added to the column in stages. After the addition of 800 µL of solution, centrifuge the column for 30-60 s and discard flow-through. Repeat until the entire solution has been added to the column membrane.
| + | |
| − | <br />
| + | |
| − | <b>Close the bag with GeneJET Purification Columns tightly after each use!</b>
| + | |
| − | </small>
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 4
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | Add a <b>700 µL</b> of <b>Wash Buffer</b> (diluted with the ethanol as described on p.3) to the GeneJET purification column. Centrifuge for 30-60 s.
| + | |
| − | <br /> Discard the flow-through and place the purification column back into the collection tube.
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 5
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer.
| + | |
| − | <br><small>
| + | |
| − | <b>Note.</b> This step is essential as the presence of residual ethanol in the DNA sample may inhibit subsequent reactions.
| + | |
| − | </small>
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 6
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube (not included).
| + | |
| − | <br />
| + | |
| − | Add <b>50 µL</b> of <b>Elution Buffer</b> to the center of the GeneJET purification column membrane and centrifuge for 1 min.
| + | |
| − | <br />
| + | |
| − | <small>
| + | |
| − | <b>Note.</b>
| + | |
| − | </small>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | <small>For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 µL does not significantly reduce the DNA yield. However, elution volumes less than 10 µL are not recommended.</small>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <small>If DNA fragment is > 10 kb, prewarm Elution Buffer to 65 °C before applying to column.</small>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | <small>If the elution volume is 10 µL and DNA amount is ≥5 µg, incubate column for 1 min at room temperature before centrifugation.</small>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 7
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | Discard the GeneJET purification column and store the purified DNA at -20 °C.
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div id="GeneJET-Plasmid-Miniprep-Kit" class="protocol">
| + | |
| − | <h3>GeneJET Plasmid Miniprep Kit</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <h5>#K0502, #K0503</h5>
| + | |
| − | <p class="author">Thermo SCIENTIFIC Product Information</p>
| + | |
| − | <p>
| + | |
| − | <h3>
| + | |
| − | Growth of Bacterial Cultures
| + | |
| − | </h3>
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | Pick a single colony from a freshly streaked selective plate to inoculate 1-5 mL of LB medium supplemented with the appropriate selection antibiotic. Incubate for 12-16 hours at 37 °C while shaking at 200-250 rpm. Use a tube or flask with a volume of least 4 times the culture volume.
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Havest the bacterial culture by centrifugation at 8000 rpm (6800 x g) in a microcentrifuge for 2 min at room temperature. Decant the supernetant and remove all remaining medium.
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | Do not overload the column:
| + | |
| − | <p>
| + | |
| − | For <b>high-copy-number plasmids</b> (<i>see</i> Table 1), do not process more than <b>5 mL</b> of bacterial culture. If more than 5 mL of such a culture are processed, the GeneJET spin column capacity (20 μL of dsDNA) will be exceeded and no increase in plasmid yield will be obtained.
| + | |
| − | <br />
| + | |
| − | For <b>low-copy-number plasmids</b> (<i>see</i> Table 1), it may be necessary to process larger volumes of bacterial culture (up to <b>10 mL</b>) to recover a sufficient quantity of DNA.
| + | |
| − | </p>
| + | |
| − | <p>
| + | |
| − | <b>Table 1.</b> Copy numbers of various vectors
| + | |
| − | </p>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | <b>High-copy</b>
| + | |
| − | <br />300-700 copies per cell
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <b>Low-copy</b>
| + | |
| − | <br />
| + | |
| − | 10-50 copies per cell
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <b>Very low-copy</b>
| + | |
| − | <br />up to 5 copies per cell
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | pUC vectors
| + | |
| − | <br />
| + | |
| − | pBluescript vectors
| + | |
| − | <br />pGEM vectors
| + | |
| − | <br />pTZ vectors
| + | |
| − | <br />pJET vectors
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | pBR322 and derivatives
| + | |
| − | <br />pACYC and derivatives
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | pSC101 and derivatives
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <br />
| + | |
| − | <p>
| + | |
| − | <h3>
| + | |
| − | PURIFICATION PROTOCOLS
| + | |
| − | </h3>
| + | |
| − | </p>
| + | |
| − | <p>
| + | |
| − | <b>
| + | |
| − | Note
| + | |
| − | </b>
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | Read IMPORTANT NOTES on p.3 before starting.
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | All purification steps should be carried out at <b>room temperature.</b>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | All centrifugations should be carried out in a table-top microcentrifuge at <b>>12000 x g</b> (10000-14000 rpm, depending on the rotor type).
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <p>
| + | |
| − | Use 1-5 mL of <i>E. coli</i> culture in LB media for purification of <b>higt-copy</b> plasmids.
| + | |
| − | <br />
| + | |
| − | For <b>low-copy</b> plasmids use up to 10 mL of culture.
| + | |
| − | </p>
| + | |
| − | <p>
| + | |
| − | <b>Protocol A. Plasmid DNA purification using centrifuges</b>
| + | |
| − | </p>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <th>
| + | |
| − | <b>Step</b>
| + | |
| − | </th>
| + | |
| − | <th>
| + | |
| − | <b>Procedure</b>
| + | |
| − | </th>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 1
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | Resuspend the pelleted cells in <b>250 μL of the Resuspension Solution</b>. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
| + | |
| − | <br />
| + | |
| − | <small><b>Note.</b> Ensure RNase A has been added to the Resuspension Solution (as describe on p.3)</small>
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 2
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | Add <b>
| + | |
| − | 250 μL of the Lysis Solution
| + | |
| − | </b> and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
| + | |
| − | <br />
| + | |
| − | <small><b>Note.</b> Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.</small>
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 3
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | Add <b>
| + | |
| − | 350 μL of the Neutralization Solution
| + | |
| − | </b> and mix immediately and thoroughly by inverting the tube 4-6 times.
| + | |
| − | <br />
| + | |
| − | <small>
| + | |
| − | <b>Note.</b> It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris.
| + | |
| − | <br />The neutralized bacterial lysate should become cloudy.
| + | |
| − | </small>
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 4
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 5
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white prepipitate.
| + | |
| − | <br /><b>Note. Close the bag with GeneJET Spin Columns tightly after each use!</b>
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 6
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
| + | |
| − | <br />
| + | |
| − | <small><b>Note.</b> Do not add bleach to the flow-through, see p.8 for Safety Information.</small>
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 7
| + | |
| − | <br />
| + | |
| − | <small>for EndA+ strains only</small>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | <i>Optional:</i> use this preliminary washing step only if EndA+ strains which have high level of nuclease activity are used.
| + | |
| − | <br />Wash the GeneJET spin column by adding 500 μL of Wash Solution I (#R1611, diluted)
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 8
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | Add <b>500 μL of Wash Solution</b> (diluted with ethanol prior to first use as described on p.3) to the GeneJET spin column. Centriifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 9
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | Repeat the wash procedure (step 8) using <b>500 μL of the Wash Solution</b>.
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 10
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | Discard the flow-trough and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 11
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add <b>
| + | |
| − | 50 μL of the Elution Buffer
| + | |
| − | </b> to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.
| + | |
| − | <br />
| + | |
| − | <small>
| + | |
| − | <b>Note.</b> An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%
| + | |
| − | <br />For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70 °C before applying to silica membrane.
| + | |
| − | </small>
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 12
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | Discard the column and store the purified plasmid DNA at -20 °C.
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div id="glycerol-stocks" class="protocol">
| + | |
| − | <h3>Glycerol stocks</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">AG Ignatova</p>
| + | |
| − | <p>
| + | |
| − | from liquid culture
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | 500 μL (50% Glycerol, 50% H<sub>2</sub>O)
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | 500 μL Cells
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Store at -80 °C
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div id="LB-Agar-plates" class="protocol">
| + | |
| − | <h3>LB-Agar Plates (+ AB)</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">AG Ignatova</p>
| + | |
| − | <ol>
| + | |
| − | <li>
| + | |
| − | Heat 250 mL LB-Agar
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Cool down to ca. 50°C
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Add 250 μL Chloramphenicol/ 12.5 μL Kanamycin (50 μg/mL)/ 25μL Ampicillin (80 μg/mL)
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Preparation of 20 plates under sterile conditions
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Store at 4 °C, mark as LB <i>CAmp/Kan/Amp Plates iGEM, dd.mm.yyyy</i>
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div id="ligation" class="protocol">
| + | |
| − | <h3>Ligation</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">AG Ignatova</p>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 1 μL
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | pSB-1C3 vector (23 ng)
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 1 μL/1.5 μL
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | GroEl-Insert (3.5 ng) / miRNA-Insert (3.3 ng)
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 2 μL
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | T4 Buffer
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 1 μL
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | T4 Ligase
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 5 μL
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | ddH<sub>2</sub>0 (6 μL for control)
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <p>
| + | |
| − | 15 min at room temperature, then 10 min at 65 °C
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div id="media" class="protocol">
| + | |
| − | <h3>Media</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">Tejas</p>
| + | |
| − | <p>
| + | |
| − | <h4>
| + | |
| − | Content
| + | |
| − | </h4>
| + | |
| − | </p>
| + | |
| − | LB-Medium
| + | |
| − | <br />M9Y Medium
| + | |
| − | <br />M9 minimal medium
| + | |
| − | <p>
| + | |
| − | <h4>
| + | |
| − | LB Medium
| + | |
| − | </h4>
| + | |
| − | </p>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Yeast extrakt
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 5 g/L
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Trypton
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 10 g/L
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | NaCl
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 10 g/L
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | RO-water add 1000 mL
| + | |
| − | </td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | pH 7.5
| + | |
| − | </td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <br />
| + | |
| − | <p>
| + | |
| − | autoclave: 20 min 121 °C
| + | |
| − | </p>
| + | |
| − | <br />
| + | |
| − | <p>
| + | |
| − | <h4>
| + | |
| − | M9Y Medium (with yeast extract)
| + | |
| − | </h4>
| + | |
| − | </p>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Na<sub>2</sub>HPO<sub>4</sub>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 6 g/L
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | KH<sub>2</sub>PO<sub>4</sub>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 3 g/L
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | NaCl
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 0.5 g/L
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | NH<sub>4</sub>Cl
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1 g/L
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Yeast extract
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 2.5 g/L
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | RO-water ad 1000 mL
| + | |
| − | </td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | pH 7.4
| + | |
| − | </td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td> autoclave: 20 min 121 °C</td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <br />
| + | |
| − | After autocalving add following substances:
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 1 M MgSO<sub>4</sub>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 2 mL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 20% Glucose
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 15 mL (end conc. 3 g/L)
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 0.1 M CaCl<sub>2</sub>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1 mL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <p>
| + | |
| − | Glucose and MgSO<sub>4</sub> autoclave separately, CaCl<sub>2</sub> to be steril filtered.
| + | |
| − | </p>
| + | |
| − | <br />
| + | |
| − | <p>
| + | |
| − | <h4>
| + | |
| − | M9 minimal medium
| + | |
| − | </h4>
| + | |
| − | </p>
| + | |
| − | M9 salts (part 1) 10x:
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Na<sub>2</sub>HPO<sub>4</sub>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 68 g
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | KH<sub>2</sub>PO<sub>4</sub>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 30 g
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | NaCl
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 5 g (do not add for osmotic stress experiment)
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | NH<sub>4</sub>Cl
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 10 g
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | Add water to 1 L. Adjust pH=7.4. Autoclave
| + | |
| − | <br />
| + | |
| − | M9 (part 2) 10x:
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | MgSO<sub>4</sub>
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 10 mM
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Glucose
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 30 g
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | Add water to 1 L. Filter sterilization.
| + | |
| − | <br />
| + | |
| − | <p>
| + | |
| − | CaCl<sub>2</sub> 100 mM stock solution. Filter sterilization
| + | |
| − | </p>
| + | |
| − | <p>
| + | |
| − | AA-Met mixture 250 mg/L each, pH=7.4. Filter sterilization
| + | |
| − | </p>
| + | |
| − | <br />
| + | |
| − | <p>To use:</p>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 10x M9 salts (part 1)
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1 mL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 10x M9 (part 2)
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1 mL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 5x AA
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 2 mL for stress experiment don’t add!
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | CaCl<sub>2</sub> 100 mM
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 0.1 mL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Water
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | up to 10 mL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div id="PCR-protocols" class="protocol">
| + | |
| − | <h3>PCR protocols</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">Zhang Gong</p>
| + | |
| − | <p><h3>Content</h3></p>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | PCR protocols
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Fragment amplification with Herculase
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Fragment amplification with <i>Pfu</i>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | mutagenesis
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <p>
| + | |
| − | <h3>
| + | |
| − | Fragment amplification with Herculase
| + | |
| − | </h3>
| + | |
| − | </p>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Template
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Primers (100˜200 μM)
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1 μL each
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 10x Herc buffer
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 5 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Water
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 39.5 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | dNTPs (10 mM each)
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1.5 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Herculase
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 0.5˜1 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td></td>
| + | |
| − | <td>
| + | |
| − | 50 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 95 °C
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 2 min
| + | |
| − | </td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 95 °C
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 30 sec
| + | |
| − | </td>
| + | |
| − | <td rowspan="3">
| + | |
| − | 10 cycles
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 55 °C
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 45˜60 sec
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 72 °C
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1 min/kb
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 95 °C
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 30 sec
| + | |
| − | </td>
| + | |
| − | <td rowspan="3">
| + | |
| − | 20 cycles
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 55 °C
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 45˜60 sec
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 72 °C
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1 min/kb + 10 sec/cycle
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 4 °C
| + | |
| − | </td>
| + | |
| − | <td>-</td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <p>
| + | |
| − | <h3>
| + | |
| − | Fragment amplification with <i>Pfu</i>
| + | |
| − | </h3>
| + | |
| − | </p>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Tamplate
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Primers (100˜200 μM)
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1 μL each
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 10x <i>Pfu</i> buffer
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 5 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Water
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 39.5 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | dNTPs (10 mM each)
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1.5 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | <i>Pfu</i> polymerase
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td></td>
| + | |
| − | <td>
| + | |
| − | 50 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 95 °C
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 2 min
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 95 °C
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 30 sec
| + | |
| − | </td>
| + | |
| − | <td rowspan="3">
| + | |
| − | 30 cycles
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 55 °C
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 45˜60 sec
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 72 °C
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 2 min/kb + 1 min
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 4 °C
| + | |
| − | </td>
| + | |
| − | <td>-</td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <p><h3>Mutagenesis</h3></p>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Tamplate (1:5 dilution)
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Primers (10 μM)
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1 μL each
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 10x <i>Pfu</i> buffer
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 5 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Water
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 39.5 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | dNTPs (10 mM each)
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1.5 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | <i>Pfu</i> polymerase
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td></td>
| + | |
| − | <td>
| + | |
| − | 50 μL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 95 °C
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 2 min
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 95 °C
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 30 sec
| + | |
| − | </td>
| + | |
| − | <td rowspan="3">
| + | |
| − | 18 cycles
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 55 °C
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 45˜60 sec
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 68 °C
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 2 min/kb + 1 min
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 4 °C
| + | |
| − | </td>
| + | |
| − | <td>-</td>
| + | |
| − | <td></td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | DpnI 1 μl, 37 °C 1 h. Then transform 5 μL into DH5α.
| + | |
| − | <p>
| + | |
| − | <b>Note:</b>
| + | |
| − | <ol>
| + | |
| − | <li>
| + | |
| − | If you get too less colonies, you may think to increase the cycles to 20, but no more! Otherwise you might get mistakes in the final sequence.
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | You can try both <i>Pfu</i> and Herculase to perform muatgenesis. <i>Pfu</i> will give less yield but more fidelity, so you should preferably use <i>Pfu</i>. If you cannot succeed with <i>Pfu</i> then you can consider trying Herculase. Just keep the same protocol!
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div id="pick-colony" class="protocol">
| + | |
| − | <h3>Pick colony</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">AG Ignatova</p>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | Inoculate each picked colony into 5 mL of liquid LB-(CAmp/ respective antibioticum)-medium, respectively
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Tubes in the 37 °C incubation room, on rotator!
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div id="preparation-for-sequencing" class="protocol">
| + | |
| − | <h3>Preparation for sequencing</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">AG Ignatova</p>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | Primer concentration: 100 μM → Concentration needed: 5 μM (dilute at 1:20)
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | take 50 nm of sample mix with 5 μL of (5 μM primer, only forward)
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div id="restriction" class="protocol">
| + | |
| − | <h3>Restriction</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">AG Ignatova</p>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 20 μL
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | pSB-1C3 (500 ng)
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 1 μL
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | EcoRI
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 1 μL
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | PstI
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 3 μL
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | FD Buffer
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 5 μL
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | ddH<sub>2</sub>O
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <p>
| + | |
| − | 20 minutes at 37 °C, following clean up with kit
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div id="SOC-medium" class="protocol">
| + | |
| − | <h3>SOC medium</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">Dagmar Stang</p>
| + | |
| − | <p> </p>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Bacto-Tryptone
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 2.000g
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Bacto-Hefe-Extract
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 0.500 g
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | NaCl
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 0.050 g
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | KCl
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 0.019 g
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | MgCl<sub>2</sub> * (H<sub>2</sub>O)<sub>6</sub>
| + | |
| − | <td>
| + | |
| − | 0.203 g
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | (without (H<sub>2</sub>O)<sub>6</sub> → 0.095 g)
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | Glucose
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 0.036 g
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | pH 7.0 with pH-Meter
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | → 10 mL (fridge)
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div id="TB-PIPES-buffer" class="protocol">
| + | |
| − | <h3>TB PIPES Buffer</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">AG Ignatova</p>
| + | |
| − | <p>
| + | |
| − | for 100 mL:
| + | |
| − | </p>
| + | |
| − | <table>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | KCl
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 1.865 g
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | CaCl x 2 H<sub>2</sub>O
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 0.220 g
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | 0.5 M PIPES
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 2 mL
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | pH 6.7
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | MgCl<sub>2</sub> x 2 H<sub>2</sub>0
| + | |
| − | </td>
| + | |
| − | <td>
| + | |
| − | 0.889 g
| + | |
| − | </td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>
| + | |
| − | ddH<sub>2</sub>O
| + | |
| − | </td>
| + | |
| − | <td> → 100 mL</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div id="transformation-efficiency" class="protocol">
| + | |
| − | <h3>Transformation Efficiency 'Competent Cell Test Kit'</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">iGEM HQ</p>
| + | |
| − | <p>
| + | |
| − | http://parts.igem.org/Help:Transformation_Efficiency_Kit
| + | |
| − | </p>
| + | |
| − | <ol>
| + | |
| − | <li>
| + | |
| − | spin down DNA (0.5, 5, 10, 20 & 50 pg/μL RFP Construct BBa_J04450, pSB1C3) from Competent Cell Test Kit (30 sec, 10.000 rpm)
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | thaw competent cells on ice, label one 2 mL tube for each concentration + pre-chill
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | 1 μL of DNA in each tube, respectively
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | add 50 μL of competent cells to each tube, incubate for 30 min on ice
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | pre-heat (42 °C), heat shock for 1 min
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | incubate for 5 min on ice
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | add 200 μL SOC media, incubate for 2 h at 37 °C
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | pipet 20 μL on each plate (triplets), respectively
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | incubate overnight at 37 °C
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | count colonies, calculate transformation efficiency
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div id="protocols-nano">
| + | |
| − | <h2 id="header-protocols-nano">
| + | |
| − | Nanolab Protocols
| + | |
| − | </h2>
| + | |
| − | <div class="protocols-list active">
| + | |
| − | | + | |
| − | <div id="alginate-capsules" class="protocol">
| + | |
| − | <h3>Fabrication of Alginate Capsules</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">iGEM-Team TU Eindhoven</p>
| + | |
| − | Buffers and solutions:
| + | |
| − | <ul class="buffers">
| + | |
| − | <li>
| + | |
| − | <p>100mM CaCl<sub>2</sub>/BaCl<sub>2</sub> solution, 0.9% NaCl solution, 2.5% alginic acid solution</p>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | Protocol:
| + | |
| − | <h3>Preparation of 0.9% NaCl Solution</h3>
| + | |
| − | <ol>
| + | |
| − | <li>
| + | |
| − | Dissolve 0.9g NaCl in 100ml MiliQ water
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Mix/vortex so that all NaCl goes into solution
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Filter into a glass flask using a syringe with a 0.22µm filter for sterilization
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| − | <h3>Preparation of 2.5% Alginate Solution</h3>
| + | |
| − | <ol>
| + | |
| − | <li>
| + | |
| − | Dissolve 2.5g alginic acid slowly in 100ml NaCl solution. During the entire dissolving process a mixing magnet should mixing the solution carefully
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Autoclave the alginic acid solution at 121°C for 30min
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| − | <h3>Preparation of 100mM CaCl<sub>2</sub>/BaCl<sub>2</sub> solution</h3>
| + | |
| − | <ol>
| + | |
| − | <li>
| + | |
| − | Dissolve 1.1098g of CaCl<sub>2</sub>/BaCl<sub>2</sub> in 100ml MiliQ water
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Mix so that all CaCl<sub>2</sub>/BaCl<sub>2</sub> goes into solution
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Filter into a glass flask using a syringe with 0.22µm filter for sterilization
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| − | <h3>Production of Alginate Beads</h3>
| + | |
| − | <ol>
| + | |
| − | <li>
| + | |
| − | Mix 1ml bacteria in 0.9% NaCl solution with 4ml alginic acid solution
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Put suspension in a sterile syringe and use it to produce droplets in 100ml of the 100mM CaCl<sub>2</sub>/BaCl<sub>2</sub> solution or use the microfluidic device
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Put the alginate beads over a filter into a falcon tube for storage
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div id="chrome-mask" class="protocol">
| + | |
| − | <h3>Fabrication of a Chrome-Mask</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">xxxxxx</p>
| + | |
| − | Protocol:
| + | |
| − | <ol>
| + | |
| − | <li>
| + | |
| − | Design structure with AutoCAD and save it as a dxf file
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Load the dxf file to the Heidelberg Laserwriter
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Clean chrome-mask with acetone in ultrasonic bath
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Spin-coat mask with photoresist S1813 at 4000rpm and 2000rpm acceleration for 1min
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Pre-bake at 90°C for 2min
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Adjust chrome-mask on the Laserwriter stage
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Turn the vacuum on with the white valves
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Convert the dxf file to a job data for the Laserwriter
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Select a proper filter and laser head
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Start exposure
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Develop mask in MF319 for 1min
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Chrome-etching for 1-3min
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Control the etching process with an optical microscope
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div id="pdms-mould" class="protocol">
| + | |
| − | <h3>Fabrication of PDMS-Devices</h3>
| + | |
| − | <div class="protocol-content active">
| + | |
| − | <p class="author">Martin Trebbin</p>
| + | |
| − | <ol>
| + | |
| − | <li>
| + | |
| − | Put pour PDMS oligomer solution (SYLGARD 184) and 1/10 (of its weight) curing agent on the SU-8 master template inside an aluminum foil coated petri dish
| + | |
| − | <ol style="list-style-type:lower-alpha;">
| + | |
| − | <li>Stir well, remove big bubbles with a pipette tip an then expose inside a desiccator for 1-2h</li>
| + | |
| − | <li>Cure in oven at 75°C for 2h</li>
| + | |
| − | </ol>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Cutting
| + | |
| − | <ol style="list-style-type:lower-alpha;">
| + | |
| − | <li>Rough cut</li>
| + | |
| − | <li>Fine cut</li>
| + | |
| − | </ol>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Drill holes with a biops-cutter with a 0.75mm needle diameter
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Clean PDMS pieces with isopropanol
| + | |
| − | <ol style="list-style-type:lower-alpha;">
| + | |
| − | <li>Rinse isopropanol on the pieces, gently rub the surface with a finger</li>
| + | |
| − | <li>Dry with air jet</li>
| + | |
| − | <li>Leave the cleaned pieces in a fume hood to dry in ambient atmosphere</li>
| + | |
| − | </ol>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Activate PDMS surface with the low pressure plasma chamber (13.56MHz, 10Watt)
| + | |
| − | <ol style="list-style-type:lower-alpha;">
| + | |
| − | <li>Power on</li>
| + | |
| − | <li>Ventilation of the chamber</li>
| + | |
| − | <li>Open hatch carefully and insert sample in the middle of the chamber</li>
| + | |
| − | <li>Turn ventilation off</li>
| + | |
| − | <li>Turn pump on</li>
| + | |
| − | <li>Open gas valve</li>
| + | |
| − | <li>Wait until pressure sets stable at 0.38mbar</li>
| + | |
| − | <li>Set power up to 10Watt and time to 100s</li>
| + | |
| − | <li>Press generate</li>
| + | |
| − | <li>Turn the pump off and the ventilation on</li>
| + | |
| − | <li>Open hatch and remove sample with gloves</li>
| + | |
| − | </ol>
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Attach the bottom/up PDMS pieces onto another
| + | |
| − | <ol style="list-style-type:lower-alpha;">
| + | |
| − | <li>Drop filtered Milipore water on the bottom</li>
| + | |
| − | <li>Put the upper part (with the holes) on the bottom part</li>
| + | |
| − | <li>Adjust the two pieces with the help of an optical microscope</li>
| + | |
| − | </ol>
| + | |
| − | </li>
| + | |
| − | | + | |
| − | <li>
| + | |
| − | Attach the PDMS to glass
| + | |
| − | </li>
| + | |
| − | <li>
| + | |
| − | Dry out oven at 40°C for 2h or overnight
| + | |
| − | </li>
| + | |
| − | </ol>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| | </div> | | </div> |
| | </div> | | </div> |
