Team:Tianjin/Protocol
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</style> | </style> | ||
- | + | <li><a href=" https://2013.igem.org/Team:Tianjin" style="padding:20px 0px 15px 0px;height:30px;">Home</a> | |
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</li> | </li> | ||
- | + | <li><a href=" https://2013.igem.org/Team:Tianjin/Project" style="padding:20px 0px 15px 0px;height:30px;">Project</a> | |
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</li> | </li> | ||
- | + | <li><a href=" https://2013.igem.org/Team:Tianjin/Safety " style="padding:20px 0px 15px 0px;height:30px;">Safety</a> | |
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- | <li><a href=" https://2013.igem.org/Team:Tianjin/ | + | <li><a href=" https://2013.igem.org/Team:Tianjin/Modeling" style="padding:20px 0px 15px 0px;height:30px;">Modeling</a> |
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</li> | </li> | ||
- | <li><a href=" https://2013.igem.org/Team:Tianjin/ | + | <li><a href=" https://2013.igem.org/Team:Tianjin/Protocol" style="padding:20px 0px 15px 0px;height:30px;">Protocol</a> |
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</li> | </li> | ||
- | <li><a href=" https://2013.igem.org/Team:Tianjin/ | + | <li class="HP"><a href="https://2013.igem.org/Team:Tianjin/Human Practice" style="padding:14px 0px 13px 0px;">Human Practice</a> |
- | < | + | </li> |
- | + | <li><a href=" https://2013.igem.org/Team:Tianjin/Data" style="padding:20px 0px 15px 0px;height:30px;">Data</a> | |
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</li> | </li> | ||
- | <li><a href=" https://2013.igem.org/Team:Tianjin/ | + | <li><a href=" https://2013.igem.org/Team:Tianjin/Notebook" style="padding:20px 0px 15px 0px;height:30px;">Notebook</a> |
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- | <li><a href=" https://2013.igem.org/Team:Tianjin/ | + | <li><a href=" https://2013.igem.org/Team:Tianjin/Team" style="padding:20px 0px 15px 0px;height:30px;">Team</a> |
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</li> | </li> | ||
+ | <li class="A-C"><a href="https://2013.igem.org/Team:Tianjin/Attributions" style="padding:7px 0px 7px 0px;">Attributions<br/>&<br/>Contributions</a> | ||
+ | </li> | ||
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<style type="text/css"> | <style type="text/css"> | ||
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+ | }); | ||
+ | </script> | ||
+ | <div class="cont"> | ||
+ | <ul> | ||
+ | |||
+ | <li class="hmain" style="margin-top:20px;"> | ||
+ | <a href="#anchor01">Luria Bertani Medium</a> | ||
+ | </li> | ||
+ | <li class="hmain"> | ||
+ | <a href="#anchor02">M9 Medium</a> | ||
+ | </li> | ||
+ | <li class="hmain"> | ||
+ | <a href="#anchor03">Extracting Alkanes</a> | ||
+ | </li> | ||
+ | <li class="hmain"> | ||
+ | <a href="#anchor04">Ligation</a> | ||
+ | </li> | ||
+ | <li class="hmain" style="font-size:14px;"> | ||
+ | <a href="#anchor05">Restriction Enzyme Digestion</a> | ||
+ | </li> | ||
+ | <li class="hmain"> | ||
+ | <a href="#anchor06">Competent Cell</a> | ||
+ | </li> | ||
+ | <li class="hmain"> | ||
+ | <a href="#anchor07">DNA Agarose Gels</a> | ||
+ | </li> | ||
+ | <li class="hmain"> | ||
+ | <a href="#anchor08" style="font-size:14px;">Agarose Gel Electrophoresis</a> | ||
+ | </li> | ||
+ | <li class="hmain"> | ||
+ | <a href="#anchor09">PCR Purification</a> | ||
+ | </li> | ||
+ | <li class="hmain"> | ||
+ | <a href="#anchor10">Gel Extraction of DNA</a> | ||
+ | </li> | ||
+ | <li class="hmain"> | ||
+ | <a href="#anchor11">Error-Prone PCR</a> | ||
+ | </li> | ||
+ | <li class="hmain"> | ||
+ | <a href="#anchor12">ColonyPCR</a> | ||
+ | </li> | ||
+ | <li class="hmain"> | ||
+ | <a href="#anchor13">Fluor Spectrophotometry</a> | ||
+ | </li> | ||
- | + | <li> | |
- | + | <a href="https://2013.igem.org/Main_Page" title="Main Page"><img src="https://static.igem.org/mediawiki/2013/7/75/IGEM-logo-blue.png" width="200px" height="200px" border="none"/></a> | |
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</div> | </div> | ||
+ | |||
+ | |||
+ | </div> | ||
</div> | </div> | ||
- | < | + | |
+ | <style type="text/css"> | ||
+ | .table1{font-family:Arial, Helvetica, sans-serif;width:100%;border-collapse:collapse;} | ||
+ | .table1 td, .table1 th{font-size:14px;border:1px solid #ccc;padding:5px 10px 5px 10px;width:150px;} | ||
+ | .table1 th {font-size:14px;text-align:left;padding-top:5px;padding-bottom:4px;background-color:#A7C942;color:#ffffff;} | ||
+ | .table1 tr.alt td {color:#000000;background-color: #fafafa;} | ||
+ | </style> | ||
+ | <div class="main"> | ||
+ | <a name="anchor01" id="anchor01"></a> | ||
+ | |||
+ | </html> | ||
+ | |||
+ | =Luria Bertani Medium= | ||
+ | |||
+ | <html> | ||
+ | |||
+ | <hr /><br /> | ||
+ | <table class="table1"> | ||
+ | <tr> | ||
+ | <td>Tryptone</td> | ||
+ | <td>10g/L</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td>Yeast extract</td> | ||
+ | <td>5g/L</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>NaCl</td> | ||
+ | <td>10g/L</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td> </td> | ||
+ | <td> </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td colspan="2"><b>Solid Luria Bertani medium: Add 15g/L Agar into Luria Bertani medium</b></td> | ||
+ | |||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <a name="anchor02" id="anchor02"></a> | ||
+ | <br/> | ||
+ | </html> | ||
+ | |||
+ | =M9 Medium= | ||
+ | |||
+ | <html> | ||
+ | |||
+ | <hr /><br /> | ||
+ | <table class="table1"> | ||
+ | <tr> | ||
+ | <td>Na<sub>2</sub>HPO<sub>4</sub>·12H<sub>2</sub>O</td> | ||
+ | <td>15.1 g/L</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <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 class="alt"> | ||
+ | <td>NH<sub>4</sub>Cl</td> | ||
+ | <td>2 g/L</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>MgSO<sub>4</sub>·7H<sub>2</sub>O</td> | ||
+ | <td>0.25 g/L</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td>CaCl<sub>2</sub></td> | ||
+ | <td>11 mg/L</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>FeCl<sub>3</sub></td> | ||
+ | <td>27 mg/L</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td>ZnCl<sub>2</sub>·4H<sub>2</sub>O</td> | ||
+ | <td>2 mg/L</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Na<sub>2</sub>MoO<sub>4</sub>·2H<sub>2</sub>O</td> | ||
+ | <td>2 mg/L</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td>CuSO<sub>4</sub></td> | ||
+ | <td>1.9 mg/L</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>H<sub>3</sub>BO<sub>3</sub></td> | ||
+ | <td>0.5 mg/L</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td>Thiamine</td> | ||
+ | <td>1 mg/L</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Bis-tris</td> | ||
+ | <td>200 mmol/L</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td> </td> | ||
+ | <td> </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Glucose</td> | ||
+ | <td>18g/L</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td> </td> | ||
+ | <td> </td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <a name="anchor03" id="anchor03"></a> | ||
+ | <br/> | ||
+ | </html> | ||
+ | |||
+ | =Extracting Alkanes= | ||
+ | |||
+ | <html> | ||
+ | |||
+ | <hr /><br /> | ||
+ | |||
+ | <p>1. Obtain 500μl sample in 1.5 ml microcentrifuge tube from 100ml medium.</p> | ||
+ | <p>2. Add 500μl pure ethyl acetate into sample.</p> | ||
+ | <p>3. Extract by adding 0.5mL of EthylAcetate, shaking at max speed for 10 min.</p> | ||
+ | <p>4. Separate the water layer and EthylAcetate layer by centrifuge at 5000 rpm for 5min at 4℃</p> | ||
+ | <p>5. Remove 300-400μl of the top Ethyl Acetate layer, filtering by membrane, and transfer to a 1.5 ml microcentrifuge tube.</p> | ||
+ | <p>6. Store extracting sample at -20℃ bridge</p> | ||
+ | |||
+ | <a name="anchor04" id="anchor04"></a> | ||
+ | <br/> | ||
+ | </html> | ||
+ | |||
+ | =Ligation= | ||
+ | |||
+ | <html> | ||
+ | <hr /><br /> | ||
+ | |||
+ | <p>1. Check the concentration of DNA fragments and vector which are going to be ligated.</p> | ||
+ | <p>2. Calcμlate the amount of part A/partB and vector added, based on the fragment length. Note that a ligation using a molar ratio of 1:3-1:5 vector to inserts.</p> | ||
+ | <p>3. Add DNA/buffer and ligase together in the EP tube.<br /></p> | ||
+ | <table class="table1"> | ||
+ | <tr> | ||
+ | <td> Reaction system </td> | ||
+ | <td>10.0μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td> Part A </td> | ||
+ | <td> A.0μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td> Vector </td> | ||
+ | <td> V.0μl </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td>10x T4 Ligase Buffer </td> | ||
+ | <td>1.0μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td> T4 Ligase </td> | ||
+ | <td>0.2μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td colspan="2">Add ddH2O until the total volume is 10.0μL</td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <p>4. Mix the reaction by pipetting up and down gently and microfuge briefly.</p> | ||
+ | <p>5. Incubate at 22°C for 40 min.</p> | ||
+ | |||
+ | <a name="anchor05" id="anchor05"></a> | ||
+ | <br/> | ||
+ | </html> | ||
+ | |||
+ | =Restriction Enzyme Digestion= | ||
+ | |||
+ | <html> | ||
+ | <hr /><br /> | ||
+ | |||
+ | <p>To check if the two selected restriction enzymes can perform effective catalysis in the same solution</p> | ||
+ | <p>1. Mix DNA solution with the suitable amount of the master mix.<br /> | ||
+ | a. | ||
+ | <table class="table1"> | ||
+ | <tr> | ||
+ | <td> Reaction system </td> | ||
+ | <td>10.0μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td> DNA solution </td> | ||
+ | <td>6.0μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>10x FD Buffer </td> | ||
+ | <td>1.0μl </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td> Each restriction enzyme </td> | ||
+ | <td>0.2μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td> ddH2O </td> | ||
+ | <td>2.8μL </td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | b. | ||
+ | <table class="table1"> | ||
+ | <tr> | ||
+ | <td> Reaction system </td> | ||
+ | <td>30.0μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td> DNA solution </td> | ||
+ | <td>3.0μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>10x FD Buffer </td> | ||
+ | <td>3.0μl </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td> Each restriction enzyme </td> | ||
+ | <td>1.0μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td> ddH2O </td> | ||
+ | <td>23.0μL </td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | |||
</p> | </p> | ||
+ | |||
+ | <p>2. Pipette up and down in the EP tube.</p> | ||
+ | <p>3. Incubate: 37°C for 30-40 min</p> | ||
+ | |||
+ | <a name="anchor06" id="anchor06"></a> | ||
+ | <br/> | ||
+ | </html> | ||
+ | |||
+ | =Competent Cell (e.g. E.coli BL 21)= | ||
+ | |||
+ | <html> | ||
+ | <hr /><br /> | ||
+ | |||
+ | <p>1.Inoculate 5μl BL21(Glycerol Storage) into 3 ml LB medium for an overnight cultures at 37 ℃ with 220rpm shaking</p> | ||
+ | <p>2.Inoculate 50μl BL21 (from step 1) into 3 ml LB medium, inoculate a culture of 3ml LB medium, incubate for 2 h at 37℃,with 220rpm shaking</p> | ||
+ | |||
+ | <p>3.Harvest the bacteria cells by centrifuge at 5000 rpm in 1.5 ml microcentrifuge tube for 5min at 4℃</p> | ||
+ | <p>4.Add 1 ml pre-cool 0.1M CaCl2 solution , mix bacteria cells by pipetting solution</p> | ||
+ | <p>5. Place the microcentrifuge tube on ice for 20min</p> | ||
+ | <p>6. Repeat step3 and step 4</p> | ||
+ | <p>7. Harvest the bacteria cells by centrifuge at 5000 rpm in 1.5 ml microcentrifuge tube for 5min at 4℃</p> | ||
+ | <p>8. Add 100 μl pre-cool 0.1M CaCl2 solution , mix bacteria cells by pipetting solution</p> | ||
+ | <p>9. Place the microcentrifuge tube on ice for 20min</p> | ||
+ | <p>10. Store Competent Cell at -80℃ bridge</p> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <a name="anchor07" id="anchor07"></a> | ||
+ | <br/> | ||
+ | </html> | ||
+ | |||
+ | =DNA Agarose Gels= | ||
+ | |||
+ | <html> | ||
+ | <hr /><br /> | ||
+ | |||
+ | <p>1. Prepare a 1% weight-to-volume agarose gel(take 100ml as example)</p> | ||
+ | <p>2. Dilute stock of 50×TAE to 1×with ddH2O.</p> | ||
+ | |||
+ | <p>3. Measure 100 ml of 1×TAE buffer.</p> | ||
+ | <p>4. Transfer 1×TAE buffer to Erlenmeyer flask.</p> | ||
+ | <p>5. Weigh out enough agarose to make 1% gel. (1% of 100mL is 1.0 g)</p> | ||
+ | <p>6. Transfer agarose to Erlenmeyer flask.</p> | ||
+ | <p>7. Melt agarose in microwave, stirring every 15-20 seconds until completely melted.</p> | ||
+ | <p>8. Allow gel to cool until Erlenmeyer flask can be handled comfortably. Then add 1:20 volume ratio (5μl) GelRed Nucleic acid dye to the gel and shake the Erlenmeyer flask to dye the gel well.</p> | ||
+ | <p>9. Pour agarose into gel tray, assemble gel pouring apparatus by inserting gate into slots.</p> | ||
+ | |||
+ | |||
+ | <a name="anchor08" id="anchor08"></a> | ||
+ | <br/> | ||
+ | </html> | ||
+ | |||
+ | =Agarose Gel Electrophoresis= | ||
+ | |||
+ | <html> | ||
+ | <hr /><br /> | ||
+ | |||
+ | <p>1. Allow agarose to cool, place the gel in the apparatus rig with the wells facing the negative end (black-colored).</p> | ||
+ | <p>2. Fill the rig with 1x TAE buffer.</p> | ||
+ | |||
+ | <p>3. Load 5µL of DNA maker into lane.</p> | ||
+ | <p>4. Mix 1µL of 10x loading buffer with 10µL DNA sample, load them into lane.</p> | ||
+ | <p>5. Run at 150V for 30 min.</p> | ||
+ | <p>6. Use Gel imaging system check gel.</p> | ||
+ | <p>7. Take picture for gel.</p> | ||
+ | |||
+ | |||
+ | <a name="anchor09" id="anchor09"></a> | ||
+ | <br/> | ||
+ | </html> | ||
+ | |||
+ | =PCR Purification= | ||
+ | |||
+ | <html> | ||
+ | <hr /><br /> | ||
+ | |||
+ | <p>TIAN™quick Midi Purification Kit</p> | ||
+ | <p>1. Balance the absorption column</p> | ||
+ | |||
+ | <p>a. Put the absorption column CB2 into the collection tube. Add 500 µl of the buffer BL to the absorption column CB2 and centrifuge at 12,000 rpm for 1 min at room temperature. Discard liquid and place the column back into the same collection tube.</p> | ||
+ | <p>2. Add the buffer PB</p> | ||
+ | <p>b. Determine the appropriate volume of the PCR reaction mixture.</p> | ||
+ | <p>c. Add 5 times the volume of the buffer PB to the mixture and then mix by shaking or overtaxing the tube in increments.</p> | ||
+ | <p>3. Absorption</p> | ||
+ | <p>d. Apply the mixture to the column. For volumes greater than 800 µl, load the column and centrifuge successively, 800 μl at a time.</p> | ||
+ | <p>e. Place the column at -20℃ for 5 min then centrifuge at 12,000 rpm for 1 min at room temperature.</p> | ||
+ | <p>f. Apply the mixture in the collection tube to the column and then repeat step "e". Discard liquid and place the column back into the same collection tube.</p> | ||
+ | <p>4. Wash</p> | ||
+ | <p>g. Wash the column by adding 600μl of PW Wash Buffer diluted with absolute ethanol. Centrifuge at 12,000 rpm for 1 min at room temp.</p> | ||
+ | <p>Note: PW Wash Buffer Concentrate must be diluted with absolute ethanol before use. See label for directions. If refrigerated, PW Wash Buffer must be brought to room temperature before use.</p> | ||
+ | <p>h. Repeat step "g" with another 600μl of PW Wash Buffer diluted with absolute ethanol.</p> | ||
+ | <p>I. Discard liquid and centrifuge the empty column for 2 min at 12,000 rpm to dry the column. Do not skip this step, it is critical for the removal of ethanol from the column.</p> | ||
+ | <p>j. Place a column into a clean microcentrifuge tube. Then place the tube into the drying baker for 10min at 50℃ to dry the column matrix.</p> | ||
+ | <p>5. Elution</p> | ||
+ | <p>k. Add 50-70μl(depending on desired concentration of final product) of EB Buffer directly onto the column matrix.</p> | ||
+ | <p>l. Incubate at 50℃ for 2 min. Centrifuge for 2 min at 12,000 rpm to elute DNA. </p> | ||
+ | <p>m. Apply the mixture in the tube to the column and then repeat step "l" to yield any residual DNA.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <a name="anchor10" id="anchor10"></a> | ||
+ | <br/> | ||
+ | </html> | ||
+ | |||
+ | =Gel Extraction of DNA (Spin Column Extraction)= | ||
+ | |||
+ | <html> | ||
+ | <hr /><br /> | ||
+ | |||
+ | <p>TIANgel Midi Purification Kit</p> | ||
+ | <p>1. Excise gel slice containing DNA fragment of interest. </p> | ||
+ | |||
+ | <p>a. Gel electrophoresis fractionates DNA fragments.</p> | ||
+ | <p>b. The gel is exposed to UV to find the DNA fragments (stained by Ethidium bromide). </p> | ||
+ | <p>c. The goal DNA band is identified.</p> | ||
+ | <p>d. Physically remove the slice of gel contains the goal DNA with clean surgical blade.</p> | ||
+ | <p>2. DNA Purification</p> | ||
+ | <p>e. Determine the appropriate volume of the gel slice by weighing it in a Clean 1.5 ml microcentrifuge tube. | ||
+ | </p> | ||
+ | <p>f. Add 3 times volume of Buffer PN more than the gel slice (0.1g gel account for 100μl). </p> | ||
+ | <p>g. Incubate the mixture at 50°C for 10 min or until the gel has completely melted. </p> | ||
+ | <p>h. Mix by shaking or overtaxing the tube in increments of 2 minutes.</p> | ||
+ | <p>I. Place a TIANGEN® DNA column CA2 in a provided 2 ml collection tube. Apply 500μl Buffer BL to the TIANGEN® DNA column, and centrifuge at 12,000 rpm for 1 min at room temperature. Discard liquid and place the TIANGEN® DNA column back into the same collection tube.</p> | ||
+ | <p>j. Apply 700 μl of the DNA/agarose solution to the TIANGEN® DNA column, incubate at -25°C for 5 min and centrifuge at 12,000 rpm for 1 min at room temperature. </p> | ||
+ | <p>k. Put the liquid back into the TIANGEN® DNA column and redo j. Discard liquid and place the TIANGEN® DNA column back into the same collection tube. For volumes greater than 700 µ l, load the column and centrifuge successively, 700 μl at a time. Each TIANGEN® DNA column has a total capacity of 25μg DNA. If the expected yield is larger, divide the sample into the appropriate number of columns.</p> | ||
+ | <p>l. Add 600 μl Buffer PW diluted with absolute ethanol into the TIANGEN® DNA column and incubate at room temperature for 2 min. Centrifuge at 12,000 rpm for 1 min at room temperature to wash the column. Discard the flow-through.</p> | ||
+ | <p>Note: Buffer PW Concentrate must be diluted with absolute ethanol before use. See label for directions. If refrigerated, Buffer PW must be brought to room temperature before use.</p> | ||
+ | <p>m. Redo step l and re-use the collection tube. </p> | ||
+ | <p>n. Centrifuge the empty TIANGEN® DNA column at 12,000 rpm for 2 min to dry the column. Do not skip this step, it is critical for the removal of ethanol from the TIANGEN® DNA column. </p> | ||
+ | <p>o. Place the TANGEN® DNA column opened into a clean 1.5 ml microcentrifuge tube and incubate at 50 °C for at least 15 minute until there is no smell of ethanol.</p> | ||
+ | <p>Add 50μl Buffer EB directly into the column and incubate at 50 °C for 5 minute. Centrifuge for 2 min at 12,000 rpm to elute DNA. This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.</p> | ||
+ | |||
+ | <a name="anchor11" id="anchor11"></a> | ||
+ | <br/> | ||
+ | </html> | ||
+ | |||
+ | = Error -Prone PCR = | ||
+ | |||
+ | <html> | ||
+ | <hr /><br /> | ||
+ | |||
+ | <p>1. Make up a master mix of everything into one microcentrifuge tube.</p> | ||
+ | <table width="100%" class="table1"> | ||
+ | <tr> | ||
+ | <td> reaction system </td> | ||
+ | <td>100.0μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td>10x error Buffer(Mg<sup>-</sup>Mn<sup>-</sup>)</td> | ||
+ | <td>10.0μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>100mM MgCl<sub>2</sub></td> | ||
+ | <td>7μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td>10mM MnCl<sub>2</sub></td> | ||
+ | <td>5μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td> dNTP </td> | ||
+ | <td>8μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td> dCTP </td> | ||
+ | <td>8μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td> dTTP </td> | ||
+ | <td>8μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td> Template </td> | ||
+ | <td>1.0μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td> Forward primer </td> | ||
+ | <td>3.0μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td> Reverse primer </td> | ||
+ | <td>3.0μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td> enzyme </td> | ||
+ | <td>1μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td> ddH2O </td> | ||
+ | <td>46μL </td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | |||
+ | <p>2. Pipette up and down in the microcentrifuge tube, drain or50.0μL solution to each PCR tube.</p> | ||
+ | |||
+ | <p>3. Run the "Error-Prone PCR" program, and adjust your extension time as described below.</p> | ||
+ | <p> The "Error-Prone PCR" program</p> | ||
+ | <p> Initial denaturation: 95°C for 5min </p> | ||
+ | <p>25cycles of:</p> | ||
+ | <p>95°C for 30 min </p> | ||
+ | <p>55°C for 30min (different primers different annealing temperature)</p> | ||
+ | <p>72°C for tmin (“t”depends on the length of goal sequence, 1minper 1000bp)</p> | ||
+ | <p> Final extension: 72°C for 10 min</p> | ||
+ | |||
+ | <a name="anchor12" id="anchor12"></a> | ||
+ | <br/> | ||
+ | </html> | ||
+ | |||
+ | = ColonyPCR= | ||
+ | |||
+ | <html> | ||
+ | <hr /><br /> | ||
+ | |||
+ | <p>1. Make up a master mix of everything into one microcentrifuge tube.</p> | ||
+ | |||
+ | <table width="100%" class="table1"> | ||
+ | <tr> | ||
+ | <td> reaction system </td> | ||
+ | <td>10.0μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td> dNTP </td> | ||
+ | <td>1.0μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td> Taq Buffer </td> | ||
+ | <td>1.0μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td> up primer </td> | ||
+ | <td>0.2μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td> down primer </td> | ||
+ | <td>0.2μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr class="alt"> | ||
+ | <td> Enzyme(fast taq)</td> | ||
+ | <td>0.1μL </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td> ddH2O </td> | ||
+ | <td>7.5μL </td> | ||
+ | </tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <p>2.Run the "Simple PCR" program, and adjust your extension time as described below.</p> | ||
+ | |||
+ | <p> The "Simple PCR" program </p> | ||
+ | <p> Initial denaturation: 95°C for 5min </p> | ||
+ | <p>30cycles of:</p> | ||
+ | <p>95°C for 30 min </p> | ||
+ | <p>55°C for 30min (different primers different annealing temperature)</p> | ||
+ | <p>72°C for tmin (“t”depends on the length of goal sequence, 1minper 1000bp)</p> | ||
+ | <p> Final extension: 72°C for 10min</p> | ||
+ | |||
+ | <a name="anchor13" id="anchor13"></a> | ||
+ | <br/> | ||
+ | </html> | ||
+ | |||
+ | = Fluor Spectrophotometry= | ||
+ | |||
+ | <html> | ||
+ | <hr /><br /> | ||
+ | |||
+ | <p> 1.Obtain 1000μl sample in 1.5 ml microcentrifuge tube from a 3ml overnight cultures E.coli.</p> | ||
+ | <p> 2.Harvest the bacteria cells by centrifuge at 8000 rpm in 1.5 ml microcentrifuge tube for 2min.</p> | ||
+ | |||
+ | <p>3. Add 1ml water, mix bacteria cells by pipetting solution </p> | ||
+ | <p>4. Remove 200μL sample and add 2000μL water, measure the optical density(OD)</p> | ||
+ | <p>5. Remove μL sample and add μL water, measure the fluorescence intensity.</p> | ||
+ | <p>6. Calculate the fluorescence intensity of per OD sample, build the model</p> | ||
+ | |||
+ | |||
+ | |||
</div> | </div> | ||
- | |||
- | |||
- | |||
</div> | </div> | ||
+ | |||
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- | <a href="#top"><img border=0 src=" | + | <a href="#top"><img border=0 src="https://static.igem.org/mediawiki/2013/6/61/Top.jpg"></a> |
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</html> | </html> |
Latest revision as of 21:20, 27 September 2013