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| Line 4: |
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| | <p><br><h3>Notebook</h3><br></p> | | <p><br><h3>Notebook</h3><br></p> |
| - |
| |
| - | <h3><a name="Calcium"></a><i>E</i>. <i>coli</i> Calcium Chloride competent cell protocol</h3>
| |
| - | <h4>Preparation</h4>
| |
| - |
| |
| - | <ol>
| |
| - | <li>Inoculate a single colony into 5 mL of LB media without any antibiotics and grow overnight at 37 °C with vigorous shaking.</li>
| |
| - | <li>Inoculate 1 mL of the desired strain into 100 mL of fresh LB, use a 500 mL flask.</li>
| |
| - | <li>Incubate at 37 °C with vigorous shaking until 0.3 - 0.4 sideOD<sub>600</sub></li>
| |
| - | <li>Put the flask on ice. Pre-chill 50 mL centrifuge tubes and the centrifuge itself at 4°C.</li>
| |
| - | <li>Centrifuge 50 mL of the culture at 8,000 rpm for 5 minutes at 4 °C.</li>
| |
| - | <li>Remove the supernatant and add 10 mL of cold CaCl<sub>2 </sub>0.1 M. Vortex until the pellet is resuspended. </li>
| |
| - | <li>Incubate on ice for 30 minutes, shake the tube once in a while.</li>
| |
| - | <li>Centrifuge at 8,000 rpm for 5 minutes at 4°C. Remove the supernatant and add 2 mL of CaCl<sub>2</sub> 0.1 M. Resuspend carefully using a micropipette. Keep always on ice.</li>
| |
| - | <li>Mix the two preparations in a tube and store on ice, or use for transformation.</li>
| |
| - | </ol>
| |
| - | <p>Note: The competent cells can be stored on ice up to two weeks. </p>
| |
| - |
| |
| - |
| |
| - | <h3><a name="Rubidium"></a><i>E</i>. <i>coli</i> Rubidium Chloride competent cell protocol</h3>
| |
| - | <h4>Preparation</h4>
| |
| - |
| |
| - | <ol>
| |
| - | <li>Inoculate 5 mL of LB broth with DH5α and incubate the culture overnight at 37°C with vigorous shacking.</li>
| |
| - | <li>Use small culture to inoculate 100 mL of LB broth. Incubate at 37°C with shacking until the culture reaches an optical density (OD595) 0.4-0.6.</li>
| |
| - | <li>Transfer to two 50 mL centrifuge tubes.</li>
| |
| - | <li>Spin at maximum speed for 5 minutes at 4°C.</li>
| |
| - | <li>Remove supernatant.</li>
| |
| - | <li>Add 20 mL of TBF1 and resuspend the pellet.</li>
| |
| - | <li>Incubate on ice for 20 minutes.</li>
| |
| - | <li>Pour off supernatant.</li>
| |
| - | <li>Centrifuge 5 minutes at 8000 rpm/4°C.</li>
| |
| - | <li>Resuspend cellular pellet with 4 mL of TBF2.</li>
| |
| - | <li>Place aliquots of 100 µL at -70°C.</li>
| |
| - | </ol>
| |
| - |
| |
| - | <h4><a name="TransformationCa"></a>
| |
| - | Heat-shock transformation of <i>Escherichia</i> <i>coli</i> competent cells </h4>
| |
| - |
| |
| - | <ol>
| |
| - | <li>Add 50 µL of Ca<sup>+2</sup> competent cells to a pre-chilled centrifuge tube. Keep always on ice until step 4.</li>
| |
| - | <li>Add plasmid DNA (100 ng) or ligation (up to 5 µL) depending on DNA concentration.</li>
| |
| - | <li>Use 1 µL of a 1 ng/µL DNA sample as positive test in a separate tube. It is recommended to use a DNA-free negative test tube as well.</li>
| |
| - | <li>Chill the tube on ice for 20 - 30 minutes.</li>
| |
| - | <li>Expose the reaction mixture to a 42ºC 1 minute heat-shock.</li>
| |
| - | <li>Put the tube on ice for 2 minutes.</li>
| |
| - | <li>Add 200 µL of antibiotic-free LB media.</li>
| |
| - | <li>Incubate at 37ºC for 20 - 30 minutes.</li>
| |
| - | <li>Spread the appropriate quantity of cells (50-200 µL) on selective LB agar plates.</li>
| |
| - | <li>Incubate overnight at 37º C.</li>
| |
| - | <li>The positive plate must have around 1,000 colonies as an optimal (1X10<sup>6</sup> transformants per µg supercoiled DNA).</li>
| |
| - | </ol>
| |
| - | <p>Notes:</p>
| |
| - | <p>Until heat-shock, handle the tubes from the upper part to avoid warming the cells. Low temperature is critical for successful transformation.</p>
| |
| - | <p>Avoid transforming with more than 5 µL of ligation mixture, as ligation buffer may reduce transformation efficiency.</p>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <h3>
| |
| - | <a name="Electrocompetent"></a>Electrocompetent <i>E. coli </i>cells preparation</b>
| |
| - |
| |
| - | <h4>
| |
| - | Preparation</h4>
| |
| - | <ol>
| |
| - | <li>Inoculate a single colony of <i>E. coli </i>in 5 mL of LB media. Grow overnight or for 5 hours at 37°C with shaking at 250 rpm.</li>
| |
| - | <li>Inoculate 2.5 mL of the previous culture in 200 mL of LB media in a 2 L flask. Grow at 37 °C shaking at 300 rpm until the culture reaches an OD of 0.5 - 0.7. </li>
| |
| - | <li>Chill the cells on ice for 10 - 15 minutes and then transfer the cells into a pre-chilled centrifuge bottle.</li>
| |
| - | <li>Centrifuge at 4,200 rpm for 10 minutes at 2 °C (Beckman J-6M).</li>
| |
| - | <li>Remove the supernatant and resuspend the pellet in 5 mL of cold water. Add 200 mL of cold water and mix well. Centrifuge at 4,200 rpm for 10 minutes at 2 °C.</li>
| |
| - | <li>Remove the supernatant and resuspend the pellet by shaking gently in the remaining liquid volume. </li>
| |
| - | <li>Add 200 mL of cold water, mix well and centrifuge at 4,200 rpm for 20 minutes at 2°C.</li>
| |
| - | <li>Add 20 mL of 10% cold glycerol and mix well. Centrifuge at 4,200 rpm for 20 minutes at 2 °C.</li>
| |
| - | <li>Add 10 mL of 10% cold glycerol to each tube. Resuspend and gather all the content of the tubes in a single tube, centrifuge and remove the supernatant.</li>
| |
| - | <li> Estimate the pellet volume and add an equal volume of 10% cold glycerol. Resuspend the cells.</li>
| |
| - | <li>Divide the final volume into pre chilled tubes (100 μl) and store at -80 °C.</li>
| |
| - | </ol>
| |
| - | <p>Note: Pre-chill all the materials that will be in contact with the cells.</p>
| |
| - |
| |
| - | <h4><a name="TransformationElec"></a>Transformation competent cells of <i>Escherichia</i> <i>coli </i>by Electroporation</h4>
| |
| - |
| |
| - |
| |
| - | <ol>
| |
| - | <li>Take a tube with 50 µL of electrocompetent <i>E</i>. <i>coli</i> cells, thaw on ice.</li>
| |
| - | <li>Add a volume containing 100 ng of DNA.</li>
| |
| - | <li>Carefully transfer the cell/DNA mix into a pre-chilled electroporation cuvette. Make sure to deposit the cells at the bottom and not to introduce any air bubbles.</li>
| |
| - | <li>Electroporate under the following conditions:</li>
| |
| - | <li>Immediately add 250 µL of SOC media to the cuvette.</li>
| |
| - | <li>Incubate with vigorous shaking (250 rpm) at 37 °C for 1 hour.</li>
| |
| - | <li>Add 750 µL of LB media and mix by pipetting up and down.</li>
| |
| - | <li>Spread 200 µL of cells onto a selective LB agar plate. </li>
| |
| - | </ol>
| |
| - | <p>Note: All must be performed on ice. Electroporation cuvettes are previously chilled on ice. DNA and bacteria must be thawed on ice too. </p>
| |
| - |
| |
| - |
| |
| - | <a name="Miniprep"></a><h3>Mini preparation of plasmid DNA</h3>
| |
| - |
| |
| - | <ol>
| |
| - | <li>Pour 1.5 mL of the culture in a 1.5 mL microcentrifuge tube and centrifuge at 14,000 rpm for 30 seconds. Remove carefully the supernatant.</li>
| |
| - | <li>Add 200 µL of Solution I. Resuspend the pellet by using vortex briefly or by pipetting up and down. Incubate at room temperature for 5 minutes. </li>
| |
| - | <li>Add 200 µL of Solution II and mix gently by inverting and rotating the tube several times. Do not vortex. Incubate at room temperature for 5 minutes.</li>
| |
| - | <li>Add 200 µL of Solution III and mix gently by inverting and rotating the tube several times. Incubate the tube on ice for 5 minutes.</li>
| |
| - | <li>Centrifuge at 14,000 rpm for 5 minutes.</li>
| |
| - | <li>Transfer the supernatant to a fresh tube containing 1 mL of 100% ethanol.</li>
| |
| - | <li>Incubate at -20 ºC for 10 minutes. (Max. 2 h)</li>
| |
| - | <li>Centrifuge at 14,000 rpm for 10 minutes. Remove the supernatant.</li>
| |
| - | <li>Add 200 µL of 70% ethanol and vortex gently for 10 seconds.</li>
| |
| - | <li>Centrifuge at 14,000 rpm for 5 minutes. Remove the supernatant by pipetting. Aspirate off any residual supernatant.</li>
| |
| - | <li>Dry at 37ºC for 5 minutes.</li>
| |
| - | <li>Add 20 µL of H<sub>2</sub>O + 20 µg/mL of RNase. Resuspend by using vortex briefly.</li>
| |
| - | <li>Run an agarose gel (0.8%) or store at 4 ºC. </li>
| |
| - | </ol>
| |
| - |
| |
| - |
| |
| - | <h4><a name="Solutions"></a>
| |
| - | Solutions for Mini preparation of Plasmid DNA</h4>
| |
| - |
| |
| - |
| |
| - | <center>
| |
| - | <table cellspacing="0" cellpadding="0">
| |
| - | <tbody>
| |
| - | <tr class="yellow">
| |
| - | <td valign="top">
| |
| - | <p><b>Solution I (200 mL)</b></p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p><b>milliliters</b><b> or grams</b></p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>- Tris HCl 1 M (pH 8.0)</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>5 mL</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>- EDTA 0.5 M (pH 8.0)</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>4 mL</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>- Distilled H<sub>2</sub>O</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>Bring the final volume up to 200 mL</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td colspan="2" valign="top">
| |
| - | <p><br></p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr class="yellow">
| |
| - | <td valign="top">
| |
| - | <p><b>Solution II (200 mL)</b></p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p><br></p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>- NaOH 10N</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>4 mL</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>- SDS (powder)</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>2.0 gr</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>- Bidistilled H<sub>2</sub>0</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>Bring the final volume up to 200 mL</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td colspan="2" valign="top">
| |
| - | <p><br></p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr class="yellow">
| |
| - | <td valign="top">
| |
| - | <p><b>Soll III (200 mL)</b></p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p><br></p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>- Potassium acetate (CH<sub>3</sub>CO<sub>2</sub>K)</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>58.8 gr</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>- Acetic acid (CH<sub>3</sub>-COOH)</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>23.0 mL</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>- Distilled H<sub>2</sub>0</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>Bring the final volume up to 200 mL</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | </center>
| |
| - | <p>Notes:</p>
| |
| - | <p>When preparing Solution II, first add a little bidistilled water, then add NaOH and dissolve carefully SDS. Finally, bring the final volume up to 200 mL with bidistilled water. <br>
| |
| - | When preparing Solution III, first add 100 mL of H<sub>2</sub>O and then the potassium acetate. Once it has been add the acetic acid and finally bring the final volume up to 200 ml with bidistilled water. </p>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <h3><a name="Agarose"></a> Agarose Gel Electrophoresis Protocol</h3>
| |
| - | <p>To be loaded:</p>
| |
| - | <ul>
| |
| - | <li><p>DNA molecular size marker (λ <i>Pst</i>I): 2 - 3 μL</p></li>
| |
| - | <li><p>Plasmid DNA: 2 - 3 μL</p></li>
| |
| - | <li><p>Enzyme restrictions: 10 μL</p></li>
| |
| - | <li><p>PCR products: 5 μL</p></li>
| |
| - | </ul>
| |
| - |
| |
| - | <p>Procedure:</p>
| |
| - |
| |
| - | <ol>
| |
| - | <li>Prepare an agarose gel of the desired concentration (see Agarose gels section).</li>
| |
| - | <li>Add the necessary SB 1X buffer into the electrophoresis tank to cover the gel.</li>
| |
| - | <li>Load the first well with marker, and then load the DNA samples mixed with loading buffer into the wells.</li>
| |
| - | <li>Plug in the anode and cathode cables so that the DNA samples can move through the gel toward the anode.</li>
| |
| - | <li>Run the electrophoresis at 200 volts.</li>
| |
| - | <li>Wait approximately 20 - 30 minutes or until the bromophenol blue reaches the end of the gel and stop the electrophoresis.</li>
| |
| - | </ol>
| |
| - | <p>Note: DNA moves toward the positive electric field (anode, usually red cable) due to the negative charges.</p>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <h4><a name="AgaroseGel"></a>Agarose gel</h4>
| |
| - |
| |
| - | <p>Concentration for supercoiled and plasmid DNA: <b>0.8%</b></p>
| |
| - | <p>For digestion reaction fragments over 1,000 bp: <b>0.8%</b></p>
| |
| - | <p>For digestion reaction fragments below 500 bp: <b>1.5%</b></p>
| |
| - | <p><b>DNA size marker (λ + <i>Pst</i>I)</b>: Use 2 or 3 μL per gel.</p>
| |
| - | <p>Note: Not needed when running supercoiled DNA samples, like plasmid DNA.</p>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <h4><a name="SB"></a>SB buffer 20X</a></h4>
| |
| - |
| |
| - | <p>SB (Sodium Borate) electrophoresis buffer, 20X Stock:</p>
| |
| - | <ol>
| |
| - | <li>In 700 mL of distilled H2O, dissolve 8 gr of NaOH. </li>
| |
| - | <li>Weight 51 of Boric Acid and dissolve ¾ parts in the NaOH solution</li>
| |
| - | <li>Dissolve the remaining Boric Acid until the buffer reach pH 8.0. </li>
| |
| - | <li>Complete to 1 L with distilled H2O and store in a sterile flask.</li>
| |
| - | </ol>
| |
| - | <p>Note: Use SB 1X as buffer to run agarose gels up to 200 volts</p>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <h4><a name="Ethidium"></a>Ethidium Bromide Gel Staining</h4>
| |
| - |
| |
| - | <ol>
| |
| - | <li>Dilute the stock to 20 μg/mL in a special container with the gel buffer.</li>
| |
| - | <li>Put the gel into the container.</li>
| |
| - | <li>Let it stain for 3 - 5 minutes.</li>
| |
| - | <li>Take the gel out of the container and soak the stained gel in water for 5 minutes or more to clear background ethidium bromide from the gel.</li>
| |
| - | <li>View the gel under a UV light source or on a UV transilluminator.</li>
| |
| - | </ol>
| |
| - | <p>Note: If you want to use ethidium bromide, confine its use to a small area of your laboratory. Wear gloves when staining, handle stained gels, and dispose of any waste. </p>
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <h4><a name="Lambda"></a>Lambda/PstI Molecular Size Marker</h4>
| |
| - |
| |
| - |
| |
| - |
| |
| - | <center>
| |
| - | <div class = "img-holder" style="width:80px">
| |
| - | <a href="https://static.igem.org/mediawiki/2011/5/59/Lambda.jpg" rel="lightbox" title="
| |
| - | Lambda molecular size marker.">
| |
| - | <img src="https://static.igem.org/mediawiki/2011/5/59/Lambda.jpg" width="80px" height="300px" alt="And-gate" align="center">
| |
| - | </a>
| |
| - | <span class="img-holder-text"><b>Lambda molecular size marker.</b></span></div>
| |
| - | </center>
| |
| - |
| |
| - | <p>Mix:</p>
| |
| - | <ul>
| |
| - | <li>phage λ DNA (500 ng/μL) 50.0 μL</li>
| |
| - | <li><i>Pst</i>I 2.5 μL</li>
| |
| - | <li>Buffer 10X 6.0 μL</li>
| |
| - | <li>H<sub>2</sub>0 1.5 μL</li></ul>
| |
| - | <b><p>Procedure:</p></b>
| |
| - | <ol>
| |
| - | <li>Mix the ingredients listed above</li>
| |
| - | <li>Incubate at 37ºC / 45 minutes</li>
| |
| - | <li>Again add 2.5 μL <i>Pst</i>I</li>
| |
| - | <li>Incubate 37ºC / 45 minutes</li>
| |
| - | <li>Add 6.0 μL Loading buffer 6X</li>
| |
| - | </ol>
| |
| - | <p>Check on agarose gel.</p>
| |
| - | <p>Notes:</p>
| |
| - | <p>Final concentration: 0.30 μgr/μL</p>
| |
| - | <p>Final Volume: 66.0 μL</p>
| |
| - | <p><br></p>
| |
| - |
| |
| - | <h3><a name="Restriction"></a>Restriction enzyme digestion of DNA</h3>
| |
| - | <p>Mix for 1 reaction, final volume of 20 µL</p>
| |
| - | <p>Add the following to a microcentrifuge tube:</p>
| |
| - | <center>
| |
| - | <table cellspacing="0" cellpadding="0">
| |
| - | <tbody>
| |
| - | <tr class="yellow">
| |
| - | <td valign="top">
| |
| - | <p>DNA</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>2-3 µg</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>Buffer 10x</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>2.0 µL</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>Enzyme (10 U/µL)</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>0.3 µL (1 enzyme unit per µg DNA)</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>H<sub>2</sub>O</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>Until 20.0 µL</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | </center>
| |
| - | <p>Incubate the mixture at 37 °C (it may change, check enzyme specifications) for 1 - 1.5 hours.</p>
| |
| - | <p>Note: Prepare a mix when possible to minimize enzyme handling.</p>
| |
| - |
| |
| - | <h3><a name="PCR"></a>PCR</h3>
| |
| - | <center>
| |
| - | <table cellspacing="0" cellpadding="0">
| |
| - | <tbody>
| |
| - | <tr class="yellow">
| |
| - | <td>
| |
| - | <p>PCR reaction mix</p>
| |
| - | </td>
| |
| - | <td>
| |
| - | </td>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>DNA template</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>Total 100 ng (In 25 μl)</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>Buffer 10x</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>2.5 μL</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>Mg<sup>++ </sup>50 mM</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>0.75 μL</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>dNTPs 20 mM</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>0.25 μL</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>Primer Fwd 100 ng/μL</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>0.50 μL</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>Primer Rv 100 ng/μL</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>0.50 μL</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>Taq Pol 5 U/μL</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>0.25 μL</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>H<sub>2</sub>O</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>To bring the volume up to 25 μL</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | </center>
| |
| - | <p>Procedure:</p>
| |
| - | <ol>
| |
| - | <li>Add the corresponding H<sub>2</sub>O to a sterile PCR tube.</li>
| |
| - | <li>Add the rest of the components but the enzyme and DNA.</li>
| |
| - | <li>Add the enzyme, mix gently.</li>
| |
| - | <li>Add the respective DNA sample and mix gently.</li>
| |
| - | <li>Spin the tube briefly.</li>
| |
| - | <li>Place the sample in the thermocycler and start your PCR program.</li>
| |
| - | </ol>
| |
| - | <p>Notes:</p>
| |
| - | <p>Put on gloves before taking the PCR mix components out of the freezer.</p>
| |
| - | <p>DNA must be added at last because it may form complexes with Mg<sup>++ </sup>and inhibit the reaction.</p>
| |
| - | <p>When possible, make a mix with all the common components to minimize enzyme waste.</p>
| |
| - | <p><br></p>
| |
| - |
| |
| - | <h3><a name="Antibiotics"></a>Antibiotics</h3>
| |
| - | <center>
| |
| - | <table cellspacing="0" cellpadding="0">
| |
| - | <tbody>
| |
| - | <tr class="yellow">
| |
| - | <td valign="top">
| |
| - | <p><b>Antibiotic</b></p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p><b>Final concentration</b></p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p><b>Stock concentration</b></p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p><b>µL per mL </b></p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>Spectinomycin (Sp)</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>100 µg/mL</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>20 µg/µL</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>5</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>Ampicillin (Amp)</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>50 µg/mL</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>10 µg/µL</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>1</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>Kanamycin (Kan)</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>50 µg/mL</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>50 µg/µL</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>1</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>Chloramphenicol (Cm)</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>34 µg/mL</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>34 µg/µL</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>1</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | <tr>
| |
| - | <td valign="top">
| |
| - | <p>Tetracycline (Tet)</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>10 µg/mL</p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>5 µg/ µL </p>
| |
| - | </td>
| |
| - | <td valign="top">
| |
| - | <p>2</p>
| |
| - | </td>
| |
| - | </tr>
| |
| - | </tbody>
| |
| - | </table>
| |
| - | </center>
| |
| - | <p>Notes:</p>
| |
| - | <p>Always verify stock concentration, in case of unknown assume the one indicated above.</p>
| |
| - | <p>When using more than one antibiotic simultaneously use half the concentration for each antibiotic.</p>
| |
| | | | |
| | </div> | | </div> |
| Line 578: |
Line 11: |
| | <a href="https://2012.igem.org/Team:UANL_Mty-Mexico/Notebook/wetlab"><li>Wetlab</a></li> | | <a href="https://2012.igem.org/Team:UANL_Mty-Mexico/Notebook/wetlab"><li>Wetlab</a></li> |
| | <a href="https://2012.igem.org/Team:UANL_Mty-Mexico/Notebook/protocols"><li><b>Protocols</b></a></li> | | <a href="https://2012.igem.org/Team:UANL_Mty-Mexico/Notebook/protocols"><li><b>Protocols</b></a></li> |
| - | <a href="#Calcium"><li>Ca<sup>+2</sup> competent cells</a></li>
| |
| - | <a href="#Rubidium"><li>Rubidium competent cells</a></li>
| |
| - | <a href="#TransformationCa"><li>Transformation</a></li>
| |
| - | <a href="#Electrocompetent"><li>Electrocompetent cells </a></li>
| |
| - | <a href="#TransformationElec"><li>Electroporation</a></li>
| |
| - | <a href="#Miniprep"><li>Miniprep</a></li>
| |
| - | <a href="#Solutions"><li>Miniprep Solutions</a></li>
| |
| - | <a href="#Agarose"><li>Electrophoresis</a></li>
| |
| - | <a href="#AgaroseGel"><li>Agarose Gels</a></li>
| |
| - | <a href="#SB"><li>SB buffer</a></li>
| |
| - | <a href="#Ethidium"><li>Gel Staining</a></li>
| |
| - | <a href="#Lambda"><li>Molecular weight marker</a></li>
| |
| - | <a href="#Restriction"><li>Enzyme digestion</a></li>
| |
| - | <a href="#PCR"><li>PCR</a></li>
| |
| - | <a href="#Antibiotics"><li>Antibiotics</a></li>
| |
| - |
| |
| | </ul> | | </ul> |
| | </div> | | </div> |
| | </html> | | </html> |
| | {{:Team:UANL_Mty-Mexico/Templates:Footer}} | | {{:Team:UANL_Mty-Mexico/Templates:Footer}} |
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