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| − | <p><strong><span style="font-family:'微软雅黑',sans-serif; font-size:18.0pt; ">Our</span></strong> team have seven wet lab experimenters, all of whom are laboratory rookies at first. We still remembered how hard for us to spend about 3 months completing the first plasmid construction. After one year iGEM training, we are skilled in molecular cloning operation - can complete multiple plasmids construction simultaneously in 3 days at the soonest now. During this time, many experiments failures we have experienced. But we have got a lot of joy from the success of the experiments at the same time. Through the training for the whole project, we deeply understand the importance of laboratory safety and strict implementation of protocols when performing an experiment. | + | <p style=" font-size:18px;font-weight:500px;line-height:27px;text-align:justify">Our team have seven wet lab experimenters, all of whom are laboratory rookies at first. We still remembered how hard for us to spend about 3 months completing the first plasmid construction. After one year iGEM training, we are skilled in molecular cloning operation - can complete multiple plasmids construction simultaneously in 3 days at the soonest now. During this time, many experiments failures we have experienced. But we have got a lot of joy from the success of the experiments at the same time. Through the training for the whole project, we deeply understand the importance of laboratory safety and strict implementation of protocols when performing an experiment. |
| | Consequently, we would like to post our protocols detailed enough for other team to repeat our experiments. Simply click on a protocol to read its steps. The side menu to scroll to the specific protocol is convenient for you to look for. | | Consequently, we would like to post our protocols detailed enough for other team to repeat our experiments. Simply click on a protocol to read its steps. The side menu to scroll to the specific protocol is convenient for you to look for. |
| | </p> | | </p> |
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| | <div class="collapsible" id="Agarplates"> Modification of chassis </div> | | <div class="collapsible" id="Agarplates"> Modification of chassis </div> |
| | <div class="collapsibleContent"> | | <div class="collapsibleContent"> |
| − | <p class="MsoNormal"><span style="font-size:14.0pt; ">(1)Lysogeny Broth (LB) </span></p>
| + | <center> <object class="file-pdf"data="https://static.igem.org/mediawiki/2019/3/3e/T--SCUT_China--Modification_of_chassis.pdf |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Formula: </span></p>
| + | "> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">NaCl 1%</span></p>
| + | <embed src="https://static.igem.org/mediawiki/2019/3/3e/T--SCUT_China--Modification_of_chassis.pdf" type="application/pdf"> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Tryptone 1%</span></p>
| + | </object></center> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Yeast extract 0.5%</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Agar 2% (Solid)</span></p>
| + | </div> |
| − | <p class="MsoNormal"><span style="font-size:14.0pt; ">(2)Antibiotic-concentrations:</span></p>
| + | |
| − | <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="559" style="border-collapse:collapse;border:none;">
| + | |
| − | <tr style="height:14.7pt;">
| + | |
| − | <td width="115" valign="top" style="width:86.25pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.7pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Antibiotic</span></p></td>
| + | |
| − | <td width="138" valign="top" style="width:103.3pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:14.7pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Formula </span></p></td>
| + | |
| − | <td width="153" valign="top" style="width:115.0pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:14.7pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Standard Concentration</span></p></td>
| + | |
| − | <td width="153" valign="top" style="width:114.6pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:14.7pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Working Concentration</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="115" valign="top" style="width:86.25pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Ampicillin </span></p></td>
| + | |
| − | <td width="138" valign="top" style="width:103.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">1g+10mL ddH<sub>2</sub>O</span></p></td>
| + | |
| − | <td width="153" valign="top" style="width:115.0pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">100mg/mL</span></p></td>
| + | |
| − | <td width="153" valign="top" style="width:114.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">25-50</span><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">μg/mL</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="115" valign="top" style="width:86.25pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">kanamycin</span></p></td>
| + | |
| − | <td width="138" valign="top" style="width:103.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">0.1g+10mL ddH<sub>2</sub>O</span></p></td>
| + | |
| − | <td width="153" valign="top" style="width:115.0pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">10mg/mL</span></p></td>
| + | |
| − | <td width="153" valign="top" style="width:114.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">10-50</span><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">μg/mL</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="115" valign="top" style="width:86.25pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Chloramphenicol</span></p></td>
| + | |
| − | <td width="138" valign="top" style="width:103.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">0.25g+10mL ethanol</span></p></td>
| + | |
| − | <td width="153" valign="top" style="width:115.0pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">100mg/mL</span></p></td>
| + | |
| − | <td width="153" valign="top" style="width:114.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">2.5-25</span><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">μg/mL</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="115" valign="top" style="width:86.25pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Carbenicillin</span></p></td>
| + | |
| − | <td width="138" valign="top" style="width:103.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">1g+10mL ddH<sub>2</sub>O</span></p></td>
| + | |
| − | <td width="153" valign="top" style="width:115.0pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">100mg/mL</span></p></td>
| + | |
| − | <td width="153" valign="top" style="width:114.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">25-50</span><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">μg/mL</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="115" valign="top" style="width:86.25pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">Tetracycline </span></p></td>
| + | |
| − | <td width="138" valign="top" style="width:103.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">0.1g+10mL ddH<sub>2</sub>O</span></p></td>
| + | |
| − | <td width="153" valign="top" style="width:115.0pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">10mg/mL</span></p></td>
| + | |
| − | <td width="153" valign="top" style="width:114.6pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">10-50</span><span style="font-family:'微软雅黑',sans-serif; font-size:9.0pt; ">μg/mL</span></p></td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <p class="MsoNormal"> </p>
| + | |
| − | <p class="MsoNormal"><span style="font-size:14.0pt; ">(3)Filtration Sterilization</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Acrodisc 32mm syringe filters with 0.2μm Supor membrane are used to remove bacteria from liquids.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-size:14.0pt; ">(4)Culture in Incubator&in Shaker</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">In Incubator: at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">In Shaker: at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220rpm</span><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; "> </span></strong></p>
| + | |
| − | <p class="MsoNormal"><span style="font-size:14.0pt; ">(5)Glycerol Stock</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><1> Add 250</span><span style="font-family:'微软雅黑',sans-serif; ">μl 60% glycerol to 750μl bacteria solution and mix them thoroughly.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><2> Store at -80</span><span style="font-family:'微软雅黑',sans-serif; ">℃.</span><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; "> </span></strong></p>
| + | |
| − | </div>
| + | |
| | </section> | | </section> |
| | | | |
| Line 118: |
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| | <div class="collapsible" id="Competence">Verification of acid tolerant factors</div> | | <div class="collapsible" id="Competence">Verification of acid tolerant factors</div> |
| | <div class="collapsibleContent"> | | <div class="collapsibleContent"> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Add ethanol (96-100%) to Buffer PW before use, check bottle tag for the adding volume. </span></p>
| + | <center> <object class="file-pdf"data="https://static.igem.org/mediawiki/2019/a/a1/T--SCUT_China--Verification_of_acid_tolerant_factors.pdf"> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(1) Column equilibration: Place a Spin Column CP3 in a clean collection tube, and add 500 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer BL to CP3. Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and put the Spin Column CP3 back into the collection tube. (Please use freshly treated spin column). </span></p>
| + | <embed src="https://static.igem.org/mediawiki/2019/a/a1/T--SCUT_China--Verification_of_acid_tolerant_factors.pdf" type="application/pdf"> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(2) Harvest 1-5 ml bacterial cells in a microcentrifuge tube by centrifugation at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g) in a conventional, table-top microcentrifuge for 1 min at room temperature (15- 25°C), then remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained (For large volume of bacterial cells, please harvest to one tube by several centrifugation step.) </span></p>
| + | </object></center> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(3) Re-suspend the bacterial pellet in 250 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer P1 (Ensure that RNase A has been added). The bacteria should be resuspended completely by vortex or pipetting up and down until no cell clumps remain. </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: No cell clumps should be visible after resuspension of the pellet, otherwise incomplete lysis will lower yield and purity. </span></p>
| + | </div> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(4) Add 250 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer P2 and mix gently and thoroughly by inverting the tube 6-8 times. Note: Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. If the lysate is still not clear, please reduce bacterial pellet. </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(5) Add 350 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer P3 and mix immediately and gently by inverting the tube 6-8 times. The solution should become cloudy. Centrifuge for 10 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Note: To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer P3. If there is still white precipitation in the supernatant, please centrifuge again. </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(6) Transfer the supernatant from step 5 to the Spin Column CP3 (place CP3 in a collection tube) by decanting or pipetting. Centrifuge for 30-60 s at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g). Discard the flow-through and set the Spin Column CP3 back into the Collection Tube. </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(7) (Optional) Wash the Spin Column CP3 by adding 500 µl Buffer PD and centrifuge for 30-60 s at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g). Discard the flow-through and put Spin Column CP3 back to the collection tube. This step is recommended to remove trace nuclease activity when using endA<sup>+</sup> strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(8) Wash the Spin Column CP3 by adding 600 µl Buffer PW (ensure that ethanol (96%-100%) has been added) and centrifuge for 30-60 s at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g). Discard the flow-through, and put the Spin Colum CP3 back into the Collection Tube. </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(9) Repeat Step 8. </span></p> | + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(10) Centrifuge for an additional 2 min at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g) to remove residual wash Buffer PW. </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: Residual ethanol from Buffer PW may inhibit subsequent enzymatic reactions. We suggest open CP3 lid and stay at room temperature for a while to get rid of residual ethanol. </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(11) Place the Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50-100 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer EB to the center of the Spin Column CP3, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 × g). </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: If the volume of eluted buffer is less than 50 </span><span style="font-family:'微软雅黑',sans-serif; ">μl, it may affect recovery efficiency. The pH value of eluted buffer will have some influence in eluting; Buffer EB or distilled water (pH 7.0-8.5) is suggested to elute plasmid DNA. For long-term storage of DNA, eluting in Buffer EB and storing at -20°C is recommended, since DNA stored in water is subject to acid hydrolysis. Repeat step 11 to increase plasmid recovery efficiency.</span></p>
| + | |
| − | </div>
| + | |
| | </section> | | </section> |
| | | | |
| Line 139: |
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| | <div class="collapsible"> Verification of Toehold Switches</div> | | <div class="collapsible"> Verification of Toehold Switches</div> |
| | <div class="collapsibleContent"> | | <div class="collapsibleContent"> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Ensure that Buffer GD and Buffer PW have been prepared with appropriate volume of ethanol (96-100%) as indicated on the bottle and shake thoroughly. </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(1) Pipet 1-5 ml bacterial culture suspension in a centrifuge tube, centrifuging for 1 min at 10,000 rpm (~11,500 </span><span style="font-family:'微软雅黑',sans-serif; ">× g), discard supernatant as possible. </span></p>
| + | <table align="center"> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(2) Add 200 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer GA. Mix thoroughly by vortex. </span></p>
| + | <object class="file-pdf"data="https://static.igem.org/mediawiki/2019/4/44/T--SCUT_China--Verification.pdf |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: For difficult-broken Gram-positive bacteria, Step 2 could be replaced by lysozyme treatment: add 180 </span><span style="font-family:'微软雅黑',sans-serif; ">μl enzymatic lysis buffer (20 mM Tris·Cl, pH 8.0; 2 mM sodium EDTA; 1.2% Triton<sup>®</sup> X-100; lysozyme (final concentration of 20 mg/ml)). Incubate for at least 30 min at 37°C. Lysozyme should be prepared with buffer, otherwise the lysozyme is not active. If RNA-Free genomic DNA is required, add 4 μl RNase A (100 mg/ml, should be prepared by user, Cat. no. RT405-12), mix by vortex for 15 s, and incubate for 5 min at room temperature (15- 25°C). </span></p>
| + | "> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(3) Add 20 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Proteinase K. Mix thoroughly by vortex. </span></p>
| + | <embed src="https://static.igem.org/mediawiki/2019/4/44/T--SCUT_China--Verification.pdf |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(4) Add 220 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer GB to the sample, vortex for 15 s, and incubate at 70°C for 10 min to yield a homogeneous solution. Briefly centrifuge the 1.5 ml centrifuge tube to remove drops from the inside of the lid. </span></p>
| + | " type="application/pdf"> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: White precipitates may form when Buffer GB is added. They will not interfere with the procedure and will dissolve during the heat incubation at 70</span><span style="font-family:'微软雅黑',sans-serif; ">°C. If precipitates do not dissolve during heat incubation, it indicates that the cell is not completely lysed and may result in low yield of DNA and impurity in DNA. </span></p>
| + | </object> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(5) Add 220 </span><span style="font-family:'微软雅黑',sans-serif; ">μl ethanol (96-100%) to the sample, and mix thoroughly by vortex for 15 s. A white precipitate may form on addition of ethanol. Briefly centrifuge the 1.5 ml centrifuge tube to remove drops from the inside of the lid. </span></p>
| + | </table> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(6) Pipet the mixture from step 5 into the Spin Column CB3 (in a 2 ml collection tube) and centrifuge at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g) for 30 s. Discard flow-through and place the spin column into the collection tube. </span></p>
| + | </div> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(7) Add 500 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer GD (Ensure ethanol (96-100%) has been added) to Spin Column CB3, and centrifuge at 12,000 rpm (~13,400 × g) for 30 s, then discard the flow-through and place the spin column into the collection tube. </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(8) Add 600 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer PW (Ensure ethanol (96-100%) has been added) to Spin Column CB3, and centrifuge at 12,000 rpm (~13,400 × g) for 30 s. Discard the flow-through and place the spin column into the collection tube. </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(9) Repeat Step 8. </span></p> | + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(10) Centrifuge at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g) for 2 min to dry the membrane completely. Note: The residual ethanol of buffer PW may affect downstream application.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(11) Place the Spin Column CB3 in a new clean 1.5 ml centrifuge tube, and pipet 50-200 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer TE or distilled water directly to the center of the membrane. Incubate at room temperature (15-25°C) for 2-5 min, and then centrifuge for 2 min at 12,000 rpm (~13,400 × g). </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: If the volume of eluted buffer is less than 50 </span><span style="font-family:'微软雅黑',sans-serif; ">μl, it may affect recovery efficiency. What’s more, the pH value of eluted buffer will have some influence in eluting, we suggest chose buffer TE or distilled water (pH 7.0-8.5) to elute gDNA. For longterm storage of DNA, eluting in Buffer TE and storing at -20°C is recommended, since DNA stored in water is subject to acid hydrolysis.</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p>
| + | |
| − | </div>
| + | |
| | </section> | | </section> |
| | | | |
| Line 160: |
Line 103: |
| | <div class="collapsible" id="GenDNA">Improvement of T7 promoters</div> | | <div class="collapsible" id="GenDNA">Improvement of T7 promoters</div> |
| | <div class="collapsibleContent"> | | <div class="collapsibleContent"> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">All Primers used in experiments meet basic requirements below:</span></p>
| + | |
| − | <ol>
| + | <table align="center"> |
| − | <li><span style="font-family:'微软雅黑',sans-serif; ">Free 3</span><span style="font-family:'微软雅黑',sans-serif; ">’-end.</span></li>
| + | <object class="file-pdf"data="https://static.igem.org/mediawiki/2019/3/36/T--SCUT_China--Improvement_of_T7_promoters.pdf"> |
| − | <li><span style="font-family:'微软雅黑',sans-serif; ">Number of Mismatches<150.</span></li>
| + | <embed src="https://static.igem.org/mediawiki/2019/3/36/T--SCUT_China--Improvement_of_T7_promoters.pdf" type="application/pdf"> |
| − | <li><span style="font-family:'微软雅黑',sans-serif; ">55</span><span style="font-family:'微软雅黑',sans-serif; ">℃<T<sub>m</sub><72℃ </span></li>
| + | </object> |
| − | <li><span style="font-family:'微软雅黑',sans-serif; ">18 bp<Length<25 bp</span></li>
| + | </table> |
| − | <li><span style="font-family:'微软雅黑',sans-serif; ">40%<GC Content<60%</span></li>
| + | </div> |
| − | </ol>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">The Softwares Oligo7 and SnapGene are used to design primers.</span></p>
| + | |
| − | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">Especially:</span></strong></p>
| + | |
| − | <ol>
| + | |
| − | <li><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">For Gibson Assembly</span></strong></li>
| + | |
| − | <li><span style="font-family:'微软雅黑',sans-serif; ">68</span><span style="font-family:'微软雅黑',sans-serif; ">℃<Overhand T<sub>m</sub><72℃ </span></li>
| + | |
| − | <li><span style="font-family:'微软雅黑',sans-serif; ">45%<GC Content<55%</span></li>
| + | |
| − | <li><span style="font-family:'微软雅黑',sans-serif; ">The difference between T<sub>m</sub> of overhand of each primer is no more than 2</span><span style="font-family:'微软雅黑',sans-serif; ">℃.</span></li>
| + | |
| − | <li><span style="font-family:'微软雅黑',sans-serif; ">The length of part combining with targeted fragment is between 18bp and 25bp.</span></li>
| + | |
| − | <li><span style="font-family:'微软雅黑',sans-serif; ">The difference between T<sub>m</sub> of combining parts of two primers is no more than 5</span><span style="font-family:'微软雅黑',sans-serif; ">℃.</span></li>
| + | |
| − | <li><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">For sequencing</span></strong></li>
| + | |
| − | </ol> | + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><1> The length is around 20bp.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><2> The number of hairpin structures and primer dimers is less than 3 respectively. And the number of mismatches is strictly less than 100.</span></p>
| + | |
| − | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(3) For overlap PCR</span></strong></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">55</span><span style="font-family:'微软雅黑',sans-serif; ">℃<T<sub>m</sub> of each primer<60℃ </span></p>
| + | |
| − | </div>
| + | |
| | </section> | | </section> |
| | | | |
| Line 189: |
Line 115: |
| | <div class="collapsible" id="plasmidDNA">Construction of VerProS pool</div> | | <div class="collapsible" id="plasmidDNA">Construction of VerProS pool</div> |
| | <div class="collapsibleContent"> | | <div class="collapsibleContent"> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Primers preparation: Carefully hold the lyophilized primer powder and centrifuge at 12000rmp for 2min. Dilute to 20 </span><span style="font-family:'微软雅黑',sans-serif; ">μM with ddH<sub>2</sub>O.</span></p>
| + | |
| − | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(1) Q5 PCR</span></strong></p>
| + | <object class="file-pdf"data="https://static.igem.org/mediawiki/2019/0/0f/T--SCUT_China--Construction_of_VerProS_pool.pdf"> |
| − | <table class=" MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="581" style="border-collapse:collapse;border:none;"> | + | <embed src="https://static.igem.org/mediawiki/2019/0/0f/T--SCUT_China--Construction_of_VerProS_pool.pdf" type="application/pdf"> |
| − | <tr>
| + | </object> |
| − | <td width="241" colspan="2" valign="top" style="width:180.8pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><a name="_Hlk16355836"><span style="font-family:'微软雅黑',sans-serif; ">10</span></a><span style="font-family:'微软雅黑',sans-serif; ">μl Assay</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | </table> |
| − | <td width="128" valign="top" style="width:96.1pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Step</span></p></td>
| + | |
| − | <td width="117" valign="top" style="width:87.95pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Temperature (</span><span style="font-family:'微软雅黑',sans-serif; ">℃)</span></p></td>
| + | </div> |
| − | <td width="47" valign="top" style="width:35.45pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Time</span></p></td>
| + | </section> |
| − | <td width="47" valign="top" style="width:35.4pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Cycle</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">5</span><span style="font-family:'微软雅黑',sans-serif; ">×T5 Reaction Buffer</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span></p></td>
| + | |
| − | <td width="128" valign="top" style="width:96.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Initial Denaturation</span></p></td>
| + | |
| − | <td width="117" valign="top" style="width:87.95pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30 s</span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">dNTPs </span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.8</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="128" valign="top" style="width:96.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Denaturation</span></p></td>
| + | |
| − | <td width="117" valign="top" style="width:87.95pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10 s</span></p></td>
| + | |
| − | <td width="47" rowspan="3" valign="top" style="width:35.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Forward primer </span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.25</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="128" valign="top" style="width:96.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing</span></p></td>
| + | |
| − | <td width="117" valign="top" style="width:87.95pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30 s</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Reverse primer</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.25</span></p></td>
| + | |
| − | <td width="128" valign="top" style="width:96.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Elongation</span></p></td>
| + | |
| − | <td width="117" valign="top" style="width:87.95pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Template</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Dependent</span></p></td>
| + | |
| − | <td width="128" valign="top" style="width:96.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Final Elongation</span></p></td>
| + | |
| − | <td width="117" valign="top" style="width:87.95pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2 min</span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Q5 polymerase</span></p></td>
| + | |
| − | <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.1</span></p></td>
| + | |
| − | <td width="128" valign="top" style="width:96.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Hold</span></p></td>
| + | |
| − | <td width="117" valign="top" style="width:87.95pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">4</span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">∞ </span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span></p></td>
| + | |
| − | <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Dependent</span></p></td>
| + | |
| − | <td width="128" valign="top" style="width:96.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="117" valign="top" style="width:87.95pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><1> The temperature of annealing=T<sub>m</sub> of primers-5</span><span style="font-family:'微软雅黑',sans-serif; ">℃.</span> </p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><2> The final concentration of each template is 1ng/</span><span style="font-family:'微软雅黑',sans-serif; ">μl.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><3> The time of elongation depends on the length of fragments to be amplified:</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><1 kb: 15 s-30 s</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">≥1 kb: 40 s/kb</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">≥2.5 kb: 1 min/kb</span></p>
| + | |
| − | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(2) T5 PCR</span></strong></p>
| + | |
| − | <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="568" style="border-collapse:collapse;border:none;">
| + | |
| − | <tr>
| + | |
| − | <td width="241" colspan="2" valign="top" style="width:180.8pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10</span><span style="font-family:'微软雅黑',sans-serif; ">μl Assay</span></p></td>
| + | |
| − | <td width="121" valign="top" style="width:90.4pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Step</span></p></td>
| + | |
| − | <td width="110" valign="top" style="width:82.75pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Temperature (</span><span style="font-family:'微软雅黑',sans-serif; ">℃)</span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:38.65pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Time</span></p></td>
| + | |
| − | <td width="44" valign="top" style="width:33.3pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Cycle</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span><span style="font-family:'微软雅黑',sans-serif; ">×T5 Super PCR Mix (Colony)</span></p></td>
| + | |
| − | <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">5</span></p></td>
| + | |
| − | <td width="121" valign="top" style="width:90.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Initial denaturation</span></p></td>
| + | |
| − | <td width="110" valign="top" style="width:82.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:38.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s</span></p></td>
| + | |
| − | <td width="44" valign="top" style="width:33.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Forward primer </span></p></td>
| + | |
| − | <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.2</span></p></td>
| + | |
| − | <td width="121" valign="top" style="width:90.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Denaturation</span></p></td>
| + | |
| − | <td width="110" valign="top" style="width:82.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:38.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10s</span></p></td>
| + | |
| − | <td width="44" rowspan="3" valign="top" style="width:33.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">29</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Feverse primer </span></p></td>
| + | |
| − | <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.2</span></p></td>
| + | |
| − | <td width="121" valign="top" style="width:90.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing</span></p></td>
| + | |
| − | <td width="110" valign="top" style="width:82.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">T<sub>m</sub>-5</span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:38.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s</span></p></td>
| + | |
| − | </tr> | + | |
| − | <tr>
| + | |
| − | <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Template</span></p></td>
| + | |
| − | <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span></p></td>
| + | |
| − | <td width="121" valign="top" style="width:90.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Elongation</span></p></td>
| + | |
| − | <td width="110" valign="top" style="width:82.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:38.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s/kb</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span></p></td>
| + | |
| − | <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2.6</span></p></td>
| + | |
| − | <td width="121" valign="top" style="width:90.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Final elongation</span></p></td>
| + | |
| − | <td width="110" valign="top" style="width:82.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:38.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2min</span></p></td>
| + | |
| − | <td width="44" valign="top" style="width:33.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="127" valign="top" style="width:95.55pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="114" valign="top" style="width:85.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="121" valign="top" style="width:90.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Hold</span></p></td>
| + | |
| − | <td width="110" valign="top" style="width:82.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">4</span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:38.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">∞ </span></p></td>
| + | |
| − | <td width="44" valign="top" style="width:33.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(3) Overlap PCR</span></strong></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Step 1:</span></p>
| + | |
| − | <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="538" style="border-collapse:collapse;border:none;">
| + | |
| − | <tr>
| + | |
| − | <td width="179" colspan="2" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">25</span><span style="font-family:'微软雅黑',sans-serif; ">μl Assay</span></p></td>
| + | |
| − | <td width="132" valign="top" style="width:99.2pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Step</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.15pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Temperature (</span><span style="font-family:'微软雅黑',sans-serif; ">℃)</span></p></td>
| + | |
| − | <td width="57" valign="top" style="width:42.5pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Time</span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.45pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Cycle</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="93" valign="top" style="width:69.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Buffer</span></p></td>
| + | |
| − | <td width="87" valign="top" style="width:65.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">5</span></p></td>
| + | |
| − | <td width="132" valign="top" style="width:99.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Initial Denaturation</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
| + | |
| − | <td width="57" valign="top" style="width:42.5pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s</span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="93" valign="top" style="width:69.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">dNTPs</span></p></td>
| + | |
| − | <td width="87" valign="top" style="width:65.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span></p></td>
| + | |
| − | <td width="132" valign="top" style="width:99.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Denaturation</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
| + | |
| − | <td width="57" valign="top" style="width:42.5pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10s</span></p></td>
| + | |
| − | <td width="47" rowspan="3" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">5</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="93" valign="top" style="width:69.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Template 1</span></p></td>
| + | |
| − | <td width="87" valign="top" style="width:65.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Dependent</span></p></td>
| + | |
| − | <td width="132" valign="top" style="width:99.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">T<sub>m</sub>-5</span></p></td>
| + | |
| − | <td width="57" valign="top" style="width:42.5pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="93" valign="top" style="width:69.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Template 2</span></p></td>
| + | |
| − | <td width="87" valign="top" style="width:65.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Dependent</span></p></td>
| + | |
| − | <td width="132" valign="top" style="width:99.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Elongation</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
| + | |
| − | <td width="57" valign="top" style="width:42.5pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s/kb</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="93" valign="top" style="width:69.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Enzyme</span></p></td>
| + | |
| − | <td width="87" valign="top" style="width:65.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.25</span></p></td>
| + | |
| − | <td width="132" valign="top" style="width:99.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Final Elongation</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
| + | |
| − | <td width="57" valign="top" style="width:42.5pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2min</span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="93" valign="top" style="width:69.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span></p></td>
| + | |
| − | <td width="87" valign="top" style="width:65.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="132" valign="top" style="width:99.2pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Hold</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">4</span></p></td>
| + | |
| − | <td width="57" valign="top" style="width:42.5pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">∞ </span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | </table> | + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">*The final concentration of each template is 50ng/</span><span style="font-family:'微软雅黑',sans-serif; ">μl.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Step 2:</span></p>
| + | |
| − | <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="557" style="border-collapse:collapse;border:none;">
| + | |
| − | <tr>
| + | |
| − | <td width="165" colspan="2" valign="top" style="width:123.55pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">50</span><span style="font-family:'微软雅黑',sans-serif; ">μl Assay (μl)</span></p></td>
| + | |
| − | <td width="166" valign="top" style="width:124.3pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Step</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.1pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Temperature (</span><span style="font-family:'微软雅黑',sans-serif; ">℃)</span></p></td>
| + | |
| − | <td width="57" valign="top" style="width:42.55pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Time</span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.45pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Cycle</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="113" valign="top" style="width:84.8pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Buffer</span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:38.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">5</span></p></td>
| + | |
| − | <td width="166" valign="top" style="width:124.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Initial Denaturation</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
| + | |
| − | <td width="57" valign="top" style="width:42.55pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s</span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="113" valign="top" style="width:84.8pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">dNTPs</span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:38.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span></p></td>
| + | |
| − | <td width="166" valign="top" style="width:124.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Denaturation</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
| + | |
| − | <td width="57" valign="top" style="width:42.55pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10s</span></p></td>
| + | |
| − | <td width="47" rowspan="3" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">25</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="113" valign="top" style="width:84.8pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Forward primer</span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:38.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1.25</span></p></td>
| + | |
| − | <td width="166" valign="top" style="width:124.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">T<sub>m</sub>-5</span></p></td>
| + | |
| − | <td width="57" valign="top" style="width:42.55pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="113" valign="top" style="width:84.8pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Reverse primer</span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:38.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1.25</span></p></td>
| + | |
| − | <td width="166" valign="top" style="width:124.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Elongation</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
| + | |
| − | <td width="57" valign="top" style="width:42.55pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s/kb</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="113" valign="top" style="width:84.8pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Template</span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:38.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">25</span></p></td>
| + | |
| − | <td width="166" valign="top" style="width:124.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Final Elongation</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
| + | |
| − | <td width="57" valign="top" style="width:42.55pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2min</span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="113" valign="top" style="width:84.8pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Enzyme</span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:38.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.25</span></p></td>
| + | |
| − | <td width="166" valign="top" style="width:124.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Hold</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">4</span></p></td>
| + | |
| − | <td width="57" valign="top" style="width:42.55pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">∞ </span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="113" valign="top" style="width:84.8pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH2O</span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:38.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">15.25</span></p></td>
| + | |
| − | <td width="166" valign="top" style="width:124.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="57" valign="top" style="width:42.55pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(4) Colony PCR</span></strong></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><1> Pick several colonies and transfer them respectively to different grids (drew outside the bottom of the dish) of a new culture plate.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><2> Culture the bacteria for 8-10h.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><3> Add 20</span><span style="font-family:'微软雅黑',sans-serif; ">μl ddH<sub>2</sub>O to PCR tubes.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><4> Scrape appropriate amounts of bacteria cells and solve them in ddH<sub>2</sub>O.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><5> Lyse the cells at 95</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 10min.</span></p>
| + | |
| − | <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="525" style="border-collapse:collapse;border:none;">
| + | |
| − | <tr>
| + | |
| − | <td width="200" colspan="2" valign="top" style="width:150.1pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10</span><span style="font-family:'微软雅黑',sans-serif; ">μl Assay</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="94" valign="top" style="width:70.25pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Step</span></p></td>
| + | |
| − | <td width="119" valign="top" style="width:89.4pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Temperature (</span><span style="font-family:'微软雅黑',sans-serif; ">℃)</span></p></td>
| + | |
| − | <td width="63" valign="top" style="width:47.05pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Time</span></p></td>
| + | |
| − | <td width="49" valign="top" style="width:37.05pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Cycle</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="148" valign="top" style="width:111.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span><span style="font-family:'微软雅黑',sans-serif; ">×T5 Super PCR Mix (Colony)</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:39.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">5</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="94" valign="top" style="width:70.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Initial denaturation</span></p></td>
| + | |
| − | <td width="119" valign="top" style="width:89.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
| + | |
| − | <td width="63" valign="top" style="width:47.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s</span></p></td>
| + | |
| − | <td width="49" valign="top" style="width:37.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="148" valign="top" style="width:111.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Forward primer </span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:39.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.2</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="94" valign="top" style="width:70.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Denaturation</span></p></td>
| + | |
| − | <td width="119" valign="top" style="width:89.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">98</span></p></td>
| + | |
| − | <td width="63" valign="top" style="width:47.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10s</span></p></td>
| + | |
| − | <td width="49" rowspan="3" valign="top" style="width:37.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">29</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="148" valign="top" style="width:111.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Feverse primer </span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:39.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.2</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="94" valign="top" style="width:70.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing</span></p></td>
| + | |
| − | <td width="119" valign="top" style="width:89.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">T<sub>m</sub>-5</span></p></td>
| + | |
| − | <td width="63" valign="top" style="width:47.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="148" valign="top" style="width:111.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Template</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:39.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="94" valign="top" style="width:70.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Elongation</span></p></td>
| + | |
| − | <td width="119" valign="top" style="width:89.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
| + | |
| − | <td width="63" valign="top" style="width:47.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">30s/kb</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="148" valign="top" style="width:111.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:39.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2.6</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="94" valign="top" style="width:70.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Final Elongation</span></p></td>
| + | |
| − | <td width="119" valign="top" style="width:89.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">72</span></p></td>
| + | |
| − | <td width="63" valign="top" style="width:47.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2min</span></p></td>
| + | |
| − | <td width="49" valign="top" style="width:37.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="148" valign="top" style="width:111.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="52" valign="top" style="width:39.1pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
| − | <td width="94" valign="top" style="width:70.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Hold</span></p></td>
| + | |
| − | <td width="119" valign="top" style="width:89.4pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">4</span></p></td>
| + | |
| − | <td width="63" valign="top" style="width:47.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">∞ </span></p></td>
| + | |
| − | <td width="49" valign="top" style="width:37.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div></section> | + | |
| | | | |
| | <section class="popout protocols"> | | <section class="popout protocols"> |
| | <div class="collapsible">Construction of working part </div> | | <div class="collapsible">Construction of working part </div> |
| | <div class="collapsibleContent"> | | <div class="collapsibleContent"> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(1) Preparation of 1% gel</span></p>
| + | <table align="center"> |
| − | <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="396" style="border-collapse:collapse;border:none;">
| + | <object class="file-pdf"data="https://static.igem.org/mediawiki/2019/2/26/T--SCUT_China--Construction_of_working_part.pdf |
| − | <tr>
| + | "> |
| − | <td width="66" valign="top" style="width:49.4pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Size</span></p></td>
| + | <embed src="https://static.igem.org/mediawiki/2019/2/26/T--SCUT_China--Construction_of_working_part.pdf |
| − | <td width="85" valign="top" style="width:63.75pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Agarose (g)</span></p></td>
| + | " type="application/pdf"> |
| − | <td width="123" valign="top" style="width:92.15pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">×TAE buffer (ml)</span></p></td>
| + | </object> |
| − | <td width="123" valign="top" style="width:92.0pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">DNA stain (</span><span style="font-family:'微软雅黑',sans-serif; ">μl)</span></p></td>
| + | </table> |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="66" valign="top" style="width:49.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Big</span></p></td>
| + | |
| − | <td width="85" valign="top" style="width:63.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.8</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">80</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.0pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span></p></td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="66" valign="top" style="width:49.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Medium</span></p></td>
| + | |
| − | <td width="85" valign="top" style="width:63.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.4</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">40</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.0pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span></p></td> | + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td width="66" valign="top" style="width:49.4pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Small</span></p></td>
| + | |
| − | <td width="85" valign="top" style="width:63.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.2</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.15pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">20</span></p></td>
| + | |
| − | <td width="123" valign="top" style="width:92.0pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.5</span></p></td>
| + | |
| − | </tr> | + | |
| − | </table>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><1> Measure out appropriate amounts of agarose and add corresponding TAE buffer.</span></p> | + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><2> Heat the solution until it becomes clear. Add appropriate amounts of DNA stain after it cools down to about 55</span><span style="font-family:'微软雅黑',sans-serif; ">℃. </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><3> Pour the solution to the mold with a comb.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><4> Let it solidify for at least 20min.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(2) Move the comb. Put the gel with the mold in the electrophoresis chamber with TAE buffer.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(3) Add 1/10 10</span><span style="font-family:'微软雅黑',sans-serif; ">×Loading buffer to each DNA sample.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(4) Pipet samples and DNA marker to wells.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(5) Run for 20-30min at 120V.</span></p>
| + | |
| | </div> | | </div> |
| | </section> | | </section> |
| Line 545: |
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| | <div class="collapsible"> Test of VerProS system</div> | | <div class="collapsible"> Test of VerProS system</div> |
| | <div class="collapsibleContent"> | | <div class="collapsibleContent"> |
| − | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(1) Gel Extraction</span></strong></p>
| + | <table align="center"> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><1> Place the gel under the ultraviolet lamp and quickly cut off the gel containing the targeted DNA fragments. Remove excess gel as much as possible.</span></p>
| + | <object class="file-pdf"data="https://static.igem.org/mediawiki/2019/5/59/T--SCUT_China--Test_of_VerProS_system.pdf |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><2> Weigh the gel and put it in a 1.5 mL centrifuge tube. Add 100</span><span style="font-family:'微软雅黑',sans-serif; ">μL Buffer GDP every 100mg gel.</span></p>
| + | "> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Water bathe the gels at 50-55</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 7-10 minutes to dissolve them completely. During the water bath, turn upside down the tube twice to fasten dissolution. If the length of DNA fragment is no more than 100bp, add 3 times volume of Buffer GDP, and equal volume of isopropanol after the water bath.</span></p>
| + | <embed src="https://static.igem.org/mediawiki/2019/5/59/T--SCUT_China--Test_of_VerProS_system.pdf |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><3> Centrifuge briefly to collect droplets on the tube wall. Place a HiPure DNA Column in a 2mL collection tube. Transfer the solution (</span><span style="font-family:'微软雅黑',sans-serif; ">≤700μL) to the column. Centrifuge for 30-60 seconds at 12,000 × g. If the volume of solution exceeds 700μL, discard the filtrate and put the column back into the collection tube. Transfer the remaining gels to the column and centrifuge at 12000 × g for 30-60 seconds.</span></p>
| + | " type="application/pdf"> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><4> Discard the filtrate and put the column back into the collection tube. Add 300 </span><span style="font-family:'微软雅黑',sans-serif; ">μL Buffer GDP to the column. Set it still for a minute. Centrifuge for 30-60 seconds at 12,000 × g.</span></p>
| + | </object> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><5> Discard the filtrate and put the column back into the collection tube. Add 600 </span><span style="font-family:'微软雅黑',sans-serif; ">μL Buffer DW2 (has been diluted with absolute ethyl alcohol) to the column. Centrifuge for 30-60 seconds at 12,000 × g.</span></p>
| + | </table> |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><6> Repeat step <5>.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><7> Discard the filtrate and put the column back into the collection tube. Centrifuge at 12,000 x g for 2 min. Open the lid of the column and dry for 10-15 minutes in the air to remove ethanol thoroughly.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><10> Place the column into a clean 1.5mL centrifuge tube and add 7-30</span><span style="font-family:'微软雅黑',sans-serif; ">μL water (preheated to 55℃) to the center of the column membrane. Set it still for 2 minutes. Centrifuge at 12,000 × g for 1 min. </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><11> Repeat step <10> to increase yield. </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><12> Remove the column and store the DNA at -20</span><span style="font-family:'微软雅黑',sans-serif; ">℃. </span></p>
| + | |
| − | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(2) Column Purification</span></strong><span style="font-family:'微软雅黑',sans-serif; "> </span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Add ethanol (96-100%) to Buffer PW before use (see bottle label for volume). All centrifuge steps are in a conventional tabletop microcentrifuge at room temperature (15-25</span><span style="font-family:'微软雅黑',sans-serif; ">°C).</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><1> Column equilibration: add 500 Buffer BL to the Spin Column CB1 (put Spin Column CB1 into a collection tube). Centrifuge for 1 min at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g) in a table-top microcentrifuge. Discard the flow-through, and then place Spin Column CB1 back into the collection tube (please use freshly treated spin column).</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><2> Add 5 volumes of Buffer PB to 1 volume of the PCR reaction or enzymatic reaction and mix. It is not necessary to remove mineral oil or kerosene.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: For example, add 250 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer PB to 50 μl PCR reaction (not including oil).</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><3> Transfer the mixture to the Spin Column CB1, incubate at room temperature (15-25</span><span style="font-family:'微软雅黑',sans-serif; ">°C) for 2 min. Centrifuge for 30-60 s at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and then place Spin Column CB1 back into the same collection tube.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: The maximum loading volume of the column is 800 </span><span style="font-family:'微软雅黑',sans-serif; ">μl. For sample volumes greater than 800 μl simply load again.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><4> Add 600 µl Buffer PW (ensure that ethanol (96-100%) has been added) to the Spin Column CB1 and centrifuge for 30-60 s at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g). Discard the flow-through, and place Spin Column CB1 back in the same collection tube.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: If the purified DNA is used for the subsequent salt sensitive experiments, such as ligation or sequencing experiment, it is suggested to stand for 2-5 min after adding Buffer PW, and then centrifuge.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><5> Repeat step <4>.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><6> Centrifuge at 12,000 rpm (~13,400 </span><span style="font-family:'微软雅黑',sans-serif; ">× g) for 2 min to remove residual Buffer PW. Discard the flow-through, and allow the column to air dry with the cap open for several minutes to dry</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">the membrane.</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: Residual ethanol from Buffer PW may inhibit subsequent experiment (enzymatic or PCR reactions).</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><7> Place the Spin Column CB1 in a clean 1.5 ml microcentrifuge tube. Add 20-50 </span><span style="font-family:'微软雅黑',sans-serif; ">μl Buffer EB to the center of membrane, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 × g).</span></p> | + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Note: If the volume of eluted buffer is less than 20 </span><span style="font-family:'微软雅黑',sans-serif; ">μl, it may affect recovery efficiency. The pH value of eluted buffer will have big influence in eluting; distilled water (pH 7.0-8.5, adjusted with</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">NaOH) is suggested to elute plasmid DNA, pH<7.0 will decrease elution efficiency. For long-term storage of DNA, eluting in Buffer EB and storing at -20</span><span style="font-family:'微软雅黑',sans-serif; ">°C is recommended, since DNA stored in</span></p>
| + | |
| − | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">water is subject to acid hydrolysis. Repeat step 7 to increase plasmid recovery efficiency.</span></p>
| + | |
| | </div> | | </div> |
| | </section> | | </section> |
| Line 591: |
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| | | | |
| | </html> | | </html> |
| | + | {{SCUT_China/footer}} |
