Team:XMU-China/protocols

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<p class="MsoNormal" align="left" style="text-align:center;"><span class="tit" style="font-family:'Times New Roman','serif'; ">Protocols</span></p>
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    <p align="center"><span class="tit">Protocols</span></p>
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<h1><a name="#_Toc01" class="subtitle">1.General protocols</a> <span style="line-height:240%; font-family:'Times New Roman','serif'; font-size:12.0pt; font-weight:normal; "> </span></h1>
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    <p> <strong ><span class="subtitle">General protocols</span></strong><br />
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<h2><a name="#_Toc02"><span class="subsubtitle">Stock solution</span></span></a> <span style="line-height:173%; font-family:'Times New Roman','serif'; font-size:11.5pt; font-weight:normal; "> </span></h2>
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      <strong class="subtitle">Stock solution</strong><br />
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<p class="MsoNormal"><span style="font-family:'Times New Roman','serif'; ">50mg/mL  Kanamycin </span></p>
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      50mg/mL  Kanamycin <br />
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<p class="MsoNormal" style="text-indent:21.3pt;"><strong>- </strong><span style="font-family:'Times New Roman','serif'; ">0.5g  Kan, 10mL water, filter sterilize with millipore express membrane, freeze in  aliquots </span></p>
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      <strong>-</strong> 0.5g  Kan, 10mL water, filter sterilize with millipore express membrane, freeze in  aliquots <br />
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<p class="MsoNormal"><span style="font-family:'Times New Roman','serif'; ">100mg/mL  Amp </span></p>
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      100mg/mL  Amp <br />
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<p class="MsoListParagraph" style="margin-left:21.0pt;text-indent:0cm;"><span style="font-family:'Times New Roman','serif'; ">- 1g Amp,  10mL water, filter sterilize with millipore express  membrane, freeze in aliquots. </span></p>
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      - 1g Amp, 10mL water, filter sterilize with millipore express  membrane, freeze in aliquots. <br />
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<p class="MsoNormal"><span style="font-family:'Times New Roman','serif'; ">50mmol/L  Arabinose</span></p>
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      50mmol/L  Arabinose<br />
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<p class="MsoListParagraph" align="left" style="margin-left:21.0pt;text-align:left;text-indent:0cm;"><span style="font-family:'Times New Roman','serif'; ">- 0.1876g Arabinose, 25mL water, filter sterilize  with millipore express membrane.</span></p>
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      - 0.1876g Arabinose, 25mL water, filter sterilize  with millipore express membrane.</p>
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<p class="MsoListParagraph" align="left" style="margin-left:21.0pt;text-align:left;text-indent:0cm;"><span style="font-family:'Times New Roman','serif'; ">&nbsp;</span></p>
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    <p><strong class="subtitle">Preparation of Competent BL21 </strong> <br />
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<p class="Default"><a name="#_Toc03"><span class="subsubtitle"><strong>Preparation of Competent BL21</strong><strong></strong></span></a> </p>
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      - Thaw an aliquot of cells (without any  plasmid in them) on ice <br />
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<p class="Default" style="margin-top:0cm;margin-right:0cm;margin-bottom:3.35pt;margin-left:1.0cm;text-indent:-7.15pt;"><span style="font-size:10.5pt; ">- T</span><span style="font-size:10.5pt; ">haw an aliquot of cells (without any  plasmid in them) on ice </span></p>
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      - To 50 mL of sterile LB, add 100&mu;L aliquot of the thawed cells: remember, this LB does not have any  antibiotic in it, so work as aseptically as possible (i.e. autoclave all  solutions and use  sterile pipettes).<br />
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<p class="Default" style="margin-top:0cm;margin-right:0cm;margin-bottom:3.35pt;margin-left:1.0cm;text-indent:-7.15pt;"><span style="font-size:10.5pt; ">- To 50 mL of sterile LB, add 100&mu;L aliquot of the thawed cells: remember, this LB does not have any  antibiotic in it, so work as aseptically as possible (i.e. autoclave all  solutions and </span><span style="font-size:10.5pt; color:windowtext; ">use  sterile pipettes).</span></p>
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      - Grow cells in the shaker at 37℃ until they reach an  OD = 0.3-0.4 at 600nm (1cm  pathlength cell). This usually takes 1.5-2 hours.<br />
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<p class="Default" style="margin-top:0cm;margin-right:0cm;margin-bottom:3.35pt;margin-left:1.0cm;text-indent:-7.15pt;"><span style="font-size:10.5pt; ">- Grow cells in the shaker at 37℃ until they reach an  OD = 0.3-0.4 at 600nm (1cm  pathlength cell). This usually takes 1.5-2 hours.</span></p>
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      - Ice down the  LB with growing cells for 10 min.<br />
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<p class="Default" style="margin-top:0cm;margin-right:0cm;margin-bottom:3.35pt;margin-left:1.0cm;text-indent:-7.15pt;"><span style="font-size:10.5pt; color:windowtext; ">- Ice down the  LB with growing cells for 10 min.</span></p>
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      - Aliquot into  sterile 1.5mL tubes and Spin down at 6000rpm for 10 minutes at 4℃; discard supernatant and.<br />
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<p class="Default" style="margin-top:0cm;margin-right:0cm;margin-bottom:3.35pt;margin-left:1.0cm;text-indent:-7.15pt;"><span style="font-size:10.5pt; color:windowtext; ">- Aliquot into  sterile 1.5mL tubes and Spin down at 6000rpm for 10 minutes at 4℃; discard supernatant and.</span></p>
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      - Ice down sterile 100mM CaCl<code>2</code> and 100mM MgC<code>2</code>solutions during  centrifugation. <br />
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<p class="Default" style="margin-top:0cm;margin-right:0cm;margin-bottom:3.35pt;margin-left:1.0cm;text-indent:-7.15pt;"><span style="font-size:10.5pt; color:windowtext; ">- Ice down sterile 100mM CaCl<sub>2 </sub>and 100mM MgCl<sub>2</sub> solutions during  centrifugation. </span></p>
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      - Gently  resuspend each pellet in 8mL 0.1M  MgCl<code>2</code> and 2mL 0.1 M CaCl<code>2</code> and combine into 2 tubes (this should take 1-2 minutes per tube) <br />
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<p class="Default" style="margin-top:0cm;margin-right:0cm;margin-bottom:3.35pt;margin-left:1.0cm;text-indent:-7.15pt;"><span style="font-size:10.5pt; color:windowtext; ">- Gently  resuspend each pellet in 8mL 0.1M  MgCl2 and 2mL 0.1 M CaCl<sub>2</sub> and combine into 2 tubes (this should take 1-2 minutes per tube) </span></p>
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      - Centrifuge  6000rpm for 10 minutes and discard supernatant. <br />
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<p class="Default" style="margin-top:0cm;margin-right:0cm;margin-bottom:3.35pt;margin-left:1.0cm;text-indent:-7.15pt;"><span style="font-size:10.5pt; color:windowtext; ">- Centrifuge  6000rpm for 10 minutes and discard supernatant. </span></p>
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      - Resuspend  each pellet on ice in 100&mu;L 0.1M of ice cold CaCl<code>2</code> and combine into one tube </p>
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<p class="Default" style="margin-top:0cm;margin-right:0cm;margin-bottom:3.35pt;margin-left:1.0cm;text-indent:-7.15pt;"><span style="font-size:10.5pt; color:windowtext; ">- Resuspend  each pellet on ice in 100<span style="font-family:'Times New Roman','serif'; color:black; ">&mu;L</span> 0.1M of ice cold CaCl<sub>2</sub> and combine into one tube </span></p>
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    <p><strong class="subtitle">Transformation </strong> <br />
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<p class="MsoNormal">&nbsp;</p>
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      - Add 10&mu;L of DNA. Swirl gently with  pipette. <br />
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<p class="Default"><a name="#_Toc04"><span class="subsubtitle"><strong>Transformation</strong></span></a></p>
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      - Incubate tubes on ice for 20 minutes <br />
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<p class="Default" style="margin-bottom:3.55pt;text-indent:23.0pt;"><span style="font-size:10.5pt; ">- Add 10&mu;L of DNA. Swirl gently with  pipette. </span></p>
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      - Heat pulse tubes in 42℃ water bath for 30 seconds. <br />
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<p class="Default" style="margin-bottom:3.55pt;text-indent:23.0pt;"><span style="font-size:10.5pt; ">- Incubate tubes on ice for 20 minutes </span></p>
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      - Incubate on ice for 2 minutes <br />
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<p class="Default" style="margin-bottom:3.55pt;text-indent:23.0pt;"><span style="font-size:10.5pt; ">- Heat pulse tubes in 42℃ water bath for 30 seconds. </span></p>
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      - Add 790uL of LB broth to each tube and incubate for an hour at 37℃ with shaking. <br />
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<p class="Default" style="margin-bottom:3.55pt;text-indent:23.0pt;"><span style="font-size:10.5pt; ">- Incubate on ice for 2 minutes </span></p>
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      - Spread 100uL and 50uL of each culture on an  LB agar plate containing the appropriate antibiotics and incubate overnight at 37℃ (spread using beads). </p>
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<p class="Default" style="text-indent:23.0pt;"><span style="font-size:10.5pt; ">- Add 790uL of LB broth to each tube and incubate for an hour at 37℃ with shaking. </span></p>
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    <p><strong class="subtitle">Purity Plasmid Miniprep</strong> <br />
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<p class="Default" style="margin-left:1.0cm;text-indent:-7.15pt;"><span style="font-size:10.5pt; ">- </span><span style="font-size:10.5pt; ">Spread 100uL and 50uL of each culture on an  LB agar plate containing the appropriate antibiotics and incubate overnight at 37℃ (spread using beads). </span></p>
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      <strong>- </strong>Centrifuge sample in Eppendorf tube approximately 1.5 mL at a time, draining off supernatant after each spin and adding more cell solution <br />
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<p class="Default" style="margin-left:1.0cm;text-indent:-7.15pt;"><span style="font-size:10.5pt; ">&nbsp;</span></p>
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      <strong>- </strong>Resuspened the pelleted cells in 250 &mu;L of the Resuspension Solution (Mixture with Solution I and RNasa A). The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.<br />
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<h2><a name="#_Toc05" class="subsubtitle">Purity Plasmid Miniprep</a></h2>
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      <strong>- </strong>Add 250 &mu;L of the Lysis Solution (Solution II) and mix thoroughly and gently by inverting the tube 5-6 times, letting it stand for 1-2minutes at room temperature until the solution becomes viscous and slightly clear.<br />
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<p class="MsoNormal" align="left" style="margin-left:28.25pt;text-align:left;text-indent:-6.95pt;"><strong>-</strong><span style="font-family:'Times New Roman','serif'; "> </span><span style="font-family:'Times New Roman','serif'; ">Centrifuge sample in Eppendorf tube approximately 1.5 mL at a time, draining off supernatant after each spin and adding more cell solution</span></p>
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      <strong>- </strong>Add 350 &mu;L of the Neutralization Solution (Solution III) and mix immediately and thoroughly by inverting the tube 5-6 times.<br />
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<p class="MsoNormal" align="left" style="margin-left:28.25pt;text-align:left;text-indent:-6.95pt;"><strong>-</strong><span style="font-family:'Times New Roman','serif'; "> Resuspened the pelleted cells in 250 &mu;L of the Resuspension Solution (Mixture with Solution I and RNasa A). The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.</span></p>
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      - Centrifuge for 10 min at  12,000rpm to pellet cell debris.<br />
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<p class="MsoNormal" align="left" style="margin-left:28.25pt;text-align:left;text-indent:-6.95pt;"><strong>- </strong><span style="font-family:'Times New Roman','serif'; ">Add 250 &mu;L of the Lysis Solution (Solution II) and mix thoroughly and gently by inverting the tube 5-6 times, letting it stand for 1-2minutes at room temperature until the solution becomes viscous and slightly clear.</span></p>
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      - Apply the supernatant to the supplied spin column by decanting. Avoid disturbing or applying the white precipitate.<br />
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<p class="MsoNormal" align="left" style="margin-left:28.25pt;text-align:left;text-indent:-6.95pt;"><strong>- </strong><span style="font-family:'Times New Roman','serif'; ">Add 350 &mu;L of the Neutralization Solution (Solution III) and mix immediately and thoroughly by inverting the tube 5-6 times.</span></p>
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      - Centrifuge for 1 min at 12,000rpm. Discard  flow-through and place the column back into the same collection tube.<br />
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<p class="MsoNormal" style="margin-left:28.25pt;text-indent:-6.95pt;"><span style="font-family:'Times New Roman','serif'; ">- Centrifuge for 10 min at  12,000rpm to pellet cell debris.</span></p>
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      - Add 500 &mu;L of the Wash Buffer PB to the spin column. Centrifuge for 1minute at 12,000rpm and discard flow-through. Place the column back into the same collection tube.<br />
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<p class="MsoNormal" align="left" style="margin-left:28.25pt;text-align:left;text-indent:-6.95pt;"><span style="font-family:'Times New Roman','serif'; ">- Apply the supernatant to the supplied spin column by decanting. Avoid disturbing or applying the white precipitate.</span></p>
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      - Add 500 &mu;L of the Wash Buffer W to the spin column. Centrifuge for 1minute at 12,000rpm and discard the flow-through. Place the column back into the same collection tube.<br />
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<p class="MsoNormal" align="left" style="margin-left:28.25pt;text-align:left;text-indent:-6.95pt;"><span style="font-family:'Times New Roman','serif'; ">- Centrifuge for 1 min at 12,000rpm. </span><span style="font-family:'Times New Roman','serif'; ">Discard  flow-through and place the column back into the same collection tube.</span></p>
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      - Repeat the step 9 again.<br />
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<p class="MsoNormal" align="left" style="margin-left:28.25pt;text-align:left;text-indent:-6.95pt;"><span style="font-family:'Times New Roman','serif'; ">- Add 500 &mu;L of the Wash Buffer PB to the spin column. Centrifuge for 1minute at 12,000rpm and  discard flow-through. Place the column back into the same collection tube.</span></p>
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      - Discard flow-through  and centrifuge for an additional 3 min to remove residual Wash Solution. <br />
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<p class="MsoNormal" align="left" style="margin-left:28.25pt;text-align:left;text-indent:-6.95pt;"><span style="font-family:'Times New Roman','serif'; ">- Add 500 &mu;L of the Wash Buffer W to the spin column. Centrifuge for 1minute at 12,000rpm and discard the flow-through. Place the column back into the same collection tube.</span></p>
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      - Place the spin column in a clean 1.5 mL centrifuge tube, and pipet 20 &mu;L Elution Buffer TE (prewarm to 60℃) directly to the center of the column without touching the membrane. Let it stand for 2 min at room temperature and centrifuge for 1 min at 12,000rpm.<br />
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<p class="MsoNormal" align="left" style="margin-left:28.25pt;text-align:left;text-indent:-6.95pt;"><span style="font-family:'Times New Roman','serif'; ">- Repeat the step 9 again.</span></p>
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      - Discard the column and  store the purified plasmid DNA at -20&deg;C.</p>
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<p class="MsoNormal" align="left" style="margin-left:28.25pt;text-align:left;text-indent:-6.95pt;"><span style="font-family:'Times New Roman','serif'; ">- </span><span style="font-family:'Times New Roman','serif'; ">Discard flow-through  and centrifuge for an additional 3 min to remove residual Wash Solution.</span></p>
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    <p class="subtitle"><strong>Reaction system of restriction endonuclease</strong></p>
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<p class="MsoNormal" align="left" style="margin-left:28.25pt;text-align:left;text-indent:-6.95pt;"><span style="font-family:'Times New Roman','serif'; ">- Place the spin column in a clean 1.5 mL centrifuge tube, and pipet 20 &mu;L Elution Buffer TE (prewarm to 60<span style="font-family:宋体; ">℃</span>) directly to the center of the column without touching the membrane. Let it stand for 2 min at room temperature and centrifuge for 1 min at 12,000rpm.</span></p>
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    <p align="left"><img width="553" height="297" src="http://2012.igem.org/wiki/images/1/1f/The_digestion_system.png" /><img width="900" height="1" src="image003.jpg" />- System1、2、3 and 4 are used for Standard BioBrick Assembly .<br />
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<p class="MsoNormal" align="left" style="margin-left:28.25pt;text-align:left;text-indent:-6.95pt;"><span style="font-family:'Times New Roman','serif'; ">- </span><span style="font-family:'Times New Roman','serif'; ">Discard the column and  store the purified plasmid DNA at -20&deg;C.</span></p>
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       - System 5 and 6 are used for Restriction  analysis. Digestion of sample: at least 500 ng DNA / 10 &micro;L volume. Digest for 4  h at 37 &deg;C, afterwards inactivated by adding 10x loading buffer and standing  for 10 min at room temperature. </p>
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<p class="MsoNormal" style="margin-left:28.25pt;text-indent:-6.95pt;"><span style="font-family:'Times New Roman','serif'; ">&nbsp;</span></p>
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     <p><strong class="subtitle">Standard BioBrick Assembly</strong><br />
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<h2><a name="#_Toc06" class="subsubtitle">Reaction system of restriction endonuclease</a> <span style="line-height:173%; font-family:'Times New Roman','serif'; font-size:11.5pt; color:black; font-weight:normal; "> </span></h2>
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       - Digestion of insert: 2 &mu;g~5 &mu;g DNA / 100 &micro;L volume, 10x H buffer, EcoRI,  SpeI. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try avoiding staining or exposure to  ultraviolet light of the insert. <br />
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<p class="MsoNormal"><span style="font-family:'Times New Roman','serif'; font-size:11.5pt; ">&nbsp;</span></p>
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       - Digestion of vector: 2 &mu;g~5 &mu;g DNA / 100 &micro;L volume, 10 x M buffer, EcoRI,  XbaI. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try to avoid staining or exposure to  ultraviolet light of the insert.</p>
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<p class="MsoNormal" align="center" style="text-align:center;"><span style="font-family:'Raavi','sans-serif'; color:black; ">Table 1. The  digestion system</span><strong><span style="font-family:'Raavi','sans-serif'; font-size:6.5pt; color:black; "> </span></strong></p>
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    <p><strong class="subtitle">Suffix Insertion </strong><br />
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<div align="center">
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       - Digestion of insert: 2 &mu;g~5 &mu;g DNA / 100 &micro;L volume, 10x M buffer, XbaI,  PstI. Digestion and inactivation. Clean up the insert.<br />
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  <table class="MsoNormalTable" border="1" cellspacing="0" cellpadding="0" width="584" style="width:437.75pt;border-collapse:collapse;border:none;">
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       - Digestion of vector : 2 &mu;g~5 &mu;g DNA / 100 &micro;L volume, 10x H buffer, SpeI,  PstI. Digestion and inactivation. Clean up the vector.</p>
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    <tr style="height:12.5pt;">
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    <p><strong class="subtitle">Ligation </strong><br />
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      <td width="73" valign="top" style="width:55.05pt;border:solid #4BACC6 1.0pt;border-bottom:none;background:#00B0F0;padding:0cm 5.4pt 0cm 5.4pt;height:12.5pt;" bgcolor="#00B0F0"><p class="MsoNormal" style="text-indent:13.05pt;"><strong><span style="font-family:'Times New Roman','serif'; font-size:6.5pt; color:black; ">&nbsp;</span></strong></p></td>
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       - After digestion and clean-up, the next step is ligation. ligation at 16℃ for 4h or at 4℃ for 16h. Table 2 is the  system of ligation. <br />
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      <td width="151" colspan="2" valign="top" style="width:4.0cm;border:none;border-top:solid #4BACC6 1.0pt;background:#00B0F0;padding:0cm 5.4pt 0cm 5.4pt;height:12.5pt;" bgcolor="#00B0F0"><p class="MsoNormal" style="text-indent:21.1pt;"><strong><span style="font-family:'Times New Roman','serif'; font-size:10.0pt; color:black; ">Prdfix Insertion</span></strong><strong><span style="font-family:'Times New Roman','serif'; font-size:6.5pt; color:black; "> </span></strong></p></td>
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       Table 2&nbsp; Ligation system</p>
+
      <td width="170" colspan="2" valign="top" style="width:127.55pt;border:solid #4BACC6 1.0pt;border-bottom:none;background:#00B0F0;padding:0cm 5.4pt 0cm 5.4pt;height:12.5pt;" bgcolor="#00B0F0"><p class="MsoNormal" align="center" style="text-align:center;text-indent:21.1pt;"><strong><span style="font-family:'Times New Roman','serif'; font-size:10.0pt; color:black; ">Suffix Insertion</span></strong><strong><span style="font-family:'Times New Roman','serif'; font-size:6.5pt; color:black; "> </span></strong></p></td>
-
    <div align="center">
+
       <td width="189" colspan="2" valign="top" style="width:5.0cm;border-top:solid #4BACC6 1.0pt;border-left:none;border-bottom:none;border-right:solid #4BACC6 1.0pt;background:#00B0F0;padding:0cm 5.4pt 0cm 5.4pt;height:12.5pt;" bgcolor="#00B0F0"><p class="MsoNormal" align="center" style="text-align:center;text-indent:21.1pt;"><strong><span style="font-family:'Times New Roman','serif'; font-size:10.0pt; color:black; ">Restriction analysis</span></strong><strong><span style="font-family:'Times New Roman','serif'; color:black; "> </span></strong></p></td>
-
       <table border="1" cellspacing="0" cellpadding="0">
+
     </tr>
-
        <tr>
+
    <tr style="height:16.5pt;">
-
          <td width="284" valign="top"><br />
+
      <td width="73" valign="top" style="width:55.05pt;border:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:16.5pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">Components</span></strong></p></td>
-
            <strong>Components</strong></td>
+
       <td width="76" valign="top" style="width:2.0cm;border-top:solid #4BACC6 1.0pt;border-left:none;border-bottom:solid #4BACC6 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:16.5pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">EcoRI-XbaI</span></strong></p></td>
-
          <td width="284" valign="top"><p align="center">Volume/&micro;L </p></td>
+
       <td width="76" valign="top" style="width:2.0cm;border:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:16.5pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">EcoRI-SpeI</span></strong></p></td>
-
        </tr>
+
      <td width="85" valign="top" style="width:63.75pt;border-top:solid #4BACC6 1.0pt;border-left:none;border-bottom:solid #4BACC6 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:16.5pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">PstI-XbaI</span></strong></p></td>
-
        <tr>
+
      <td width="85" valign="top" style="width:63.8pt;border:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:16.5pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">PstI-SpeI</span></strong></p></td>
-
          <td width="284" valign="top"><p align="center">Digested vector </p></td>
+
       <td width="95" valign="top" style="width:70.9pt;border-top:solid #4BACC6 1.0pt;border-left:none;border-bottom:solid #4BACC6 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:16.5pt;"><p class="MsoNormal"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">Double digestion</span></strong></p></td>
-
          <td width="284" valign="top"><p align="center">7 </p></td>
+
      <td width="94" valign="top" style="width:70.85pt;border:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:16.5pt;"><p class="MsoNormal"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">Single digestion</span></strong></p></td>
-
        </tr>
+
    </tr>
-
        <tr>
+
    <tr style="height:20.2pt;">
-
          <td width="284" valign="top"><p align="center">Digested insert </p></td>
+
       <td width="73" valign="top" style="width:55.05pt;border-top:none;border-left:solid #4BACC6 1.0pt;border-bottom:none;border-right:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:20.2pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">DNA Sample/&mu;L</span></strong></p></td>
-
          <td width="284" valign="top"><p align="center">1 </p></td>
+
      <td width="76" valign="top" style="width:2.0cm;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:20.2pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">80</span></strong></p></td>
-
        </tr>
+
       <td width="76" valign="top" style="width:2.0cm;border-top:none;border-left:solid #4BACC6 1.0pt;border-bottom:none;border-right:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:20.2pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">80</span></strong></p></td>
-
        <tr>
+
       <td width="85" valign="top" style="width:63.75pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:20.2pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">80</span></strong></p></td>
-
          <td width="284" valign="top"><p align="center">10<strong>&times; T4 ligation buffer</strong></p></td>
+
      <td width="85" valign="top" style="width:63.8pt;border-top:none;border-left:solid #4BACC6 1.0pt;border-bottom:none;border-right:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:20.2pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">80</span></strong></p></td>
-
          <td width="284" valign="top"><p align="center">1 </p></td>
+
       <td width="95" valign="top" style="width:70.9pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:20.2pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">16</span></strong></p></td>
-
        </tr>
+
      <td width="94" valign="top" style="width:70.85pt;border-top:none;border-left:solid #4BACC6 1.0pt;border-bottom:none;border-right:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:20.2pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">16</span></strong></p></td>
-
        <tr>
+
    </tr>
-
          <td width="284" valign="top"><p align="center">T4 ligase </p></td>
+
    <tr style="height:20.45pt;">
-
          <td width="284" valign="top"><p align="center">1 </p></td>
+
      <td width="73" valign="top" style="width:55.05pt;border:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:20.45pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">10</span></strong><strong><span style="font-family:宋体; font-size:8.0pt; color:black; ">&times;</span></strong><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">M buffer/&mu;L</span></strong></p></td>
-
        </tr>
+
      <td width="76" valign="top" style="width:2.0cm;border-top:solid #4BACC6 1.0pt;border-left:none;border-bottom:solid #4BACC6 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:20.45pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">10</span></strong></p></td>
-
        <tr>
+
      <td width="76" valign="top" style="width:2.0cm;border:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:20.45pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:宋体; font-size:8.0pt; color:black; ">&mdash;</span></strong><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; "> </span></strong></p></td>
-
          <td width="284" valign="top"><p align="center">Total </p></td>
+
      <td width="85" valign="top" style="width:63.75pt;border-top:solid #4BACC6 1.0pt;border-left:none;border-bottom:solid #4BACC6 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:20.45pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">10</span></strong></p></td>
-
          <td width="284" valign="top"><p align="center">10 </p></td>
+
      <td width="85" valign="top" style="width:63.8pt;border:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:20.45pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
        </tr>
+
      <td width="95" valign="top" style="width:70.9pt;border-top:solid #4BACC6 1.0pt;border-left:none;border-bottom:solid #4BACC6 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:20.45pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
       </table>
+
      <td width="94" valign="top" style="width:70.85pt;border:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:20.45pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
    </div>
+
    </tr>
-
    <p>&nbsp;</p>
+
    <tr style="height:20.7pt;">
-
    <p><strong class="subtitle">Restriction analysis</strong><br />
+
      <td width="73" valign="top" style="width:55.05pt;border-top:none;border-left:solid #4BACC6 1.0pt;border-bottom:none;border-right:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:20.7pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">10</span></strong><strong><span style="font-family:宋体; font-size:8.0pt; color:black; ">&times;</span></strong><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">H buffer/&mu;L</span></strong></p></td>
-
       - Pick one colony with a sterile tip and cultivation in 20mL LB for  overnight at 37 ℃<br />
+
      <td width="76" valign="top" style="width:2.0cm;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:20.7pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
       - Isolation of Plasmid<br />
+
      <td width="76" valign="top" style="width:2.0cm;border-top:none;border-left:solid #4BACC6 1.0pt;border-bottom:none;border-right:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:20.7pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">10</span></strong></p></td>
-
       - Digest BioBrick,the system of  Restriction analysis refer to table1<br />
+
      <td width="85" valign="top" style="width:63.75pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:20.7pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
       - Gel electrophoresis:add 2.2 &micro;L loading  buffer to digestion mixture. An agarose concentration is 1 %.</p>
+
      <td width="85" valign="top" style="width:63.8pt;border-top:none;border-left:solid #4BACC6 1.0pt;border-bottom:none;border-right:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:20.7pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">10</span></strong></p></td>
-
    <p><strong class="subtitle">Gel Extraction</strong><br />
+
      <td width="95" valign="top" style="width:70.9pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:20.7pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">2.0</span></strong></p></td>
-
       - Weigh a 1.5mL centrifuge tube  for each DNA fragment to be isolated and record the weight.<br />
+
      <td width="94" valign="top" style="width:70.85pt;border-top:none;border-left:solid #4BACC6 1.0pt;border-bottom:none;border-right:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:20.7pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">2.0</span></strong></p></td>
-
       - Excise gel slice containing the  DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as  possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 Ml tube and weigh. Record the weight of the gel slice.<br />
+
    </tr>
-
       - Add Bing Buffer BD at a ratio  of 100&mu;L of solution per 100mg of agarose gel slices.<br />
+
    <tr style="height:17.25pt;">
-
       - &nbsp;Incubate the gel mixture at 55-65℃ for 7-10 min or until the gel slice  is completely dissolved. Mix the tube by inversion every few minutes to  facilitate the melting process. Ensure that the gel is completely dissolved.<br />
+
      <td width="73" valign="top" style="width:55.05pt;border:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:17.25pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">PstI/&mu;L</span></strong></p></td>
-
       - &nbsp;After the dissolved gel mixture cool  down, transfer it to the Spin Columns assembly and incubate for 2 minute at  room temperature.<br />
+
      <td width="76" valign="top" style="width:2.0cm;border-top:solid #4BACC6 1.0pt;border-left:none;border-bottom:solid #4BACC6 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:17.25pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
       - Centrifuge the Spin Columns  assembly in a microcentrifuge at 12,000 rpm for 1 minute, then discard the  flow-through.<br />
+
      <td width="76" valign="top" style="width:2.0cm;border:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:17.25pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
       - &nbsp;Wash the columns by adding 500 &mu;L of Wash  Buffer PE to the Columns. Centrifuge the columns assembly for 1 minute at 12,000  rpm, then discard the flow-through.<br />
+
       <td width="85" valign="top" style="width:63.75pt;border-top:solid #4BACC6 1.0pt;border-left:none;border-bottom:solid #4BACC6 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:17.25pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">5.0</span></strong></p></td>
-
       - &nbsp;Repeat step 7 again.<br />
+
      <td width="85" valign="top" style="width:63.8pt;border:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:17.25pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">5.0</span></strong></p></td>
-
       - Centrifuge the Columns for an  additional 3 min to completely remove residual wash buffer.<br />
+
      <td width="95" valign="top" style="width:70.9pt;border-top:solid #4BACC6 1.0pt;border-left:none;border-bottom:solid #4BACC6 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:17.25pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">1.0</span></strong></p></td>
-
       - &nbsp;Empty the Collection Tube and  recentrifuge the column assembly for 1 minute with the microcentrifuge lid open  (or off) to allow evaporation of any residual ethanol.<br />
+
       <td width="94" valign="top" style="width:70.85pt;border:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:17.25pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
       - Place the spin column in a  clean 1.5 mL microcentrifuge tube, and pipet 20 &mu;L deionized water (pH is  8.0-8.5 and prewarm to 60℃)directly to the center of the column  without touching the membrane. Incubate at room temperature for 2 min.<br />
+
    </tr>
-
       - Centrifuge for 1 minute at  12,000 rpm. Discard the columns and store the microcentrifuge tube containing  the eluted DNA at &ndash;20℃.</p>
+
    <tr style="height:13.95pt;">
-
    <p><span class="subtitle"><strong>Characterization</strong><br />
+
       <td width="73" valign="top" style="width:55.05pt;border-top:none;border-left:solid #4BACC6 1.0pt;border-bottom:none;border-right:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.95pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">XbaI/&mu;L</span></strong></p></td>
-
       <strong>Fluorescence Measurements</strong></span><br />
+
       <td width="76" valign="top" style="width:2.0cm;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.95pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">5.0</span></strong></p></td>
-
       - The samples to be tested are  cultured from plates in 20 ml of the Basal Minimal Medium with appropriate  antibiotics and incubated overnight at 37 &deg;C at 200 rpm. <br />
+
       <td width="76" valign="top" style="width:2.0cm;border-top:none;border-left:solid #4BACC6 1.0pt;border-bottom:none;border-right:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.95pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
      - The culture is checked for OD600 next day and then subculture by the same medium with antibiotics at 37 &deg;C  shaking for 2 hours.&nbsp; <br />
+
      <td width="85" valign="top" style="width:63.75pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.95pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">5.0</span></strong></p></td>
-
      - Add corresponding inducer at concentration gradients into the above-mentioned culture and keep on incubating. During the  time incubating, every 15 min, take 1 mL bacteria liquid, then centrifuge the  cells( 6000 rpm, 10 min ) and resuspend them in 1 mL PBS. At last, pipette to a 96 well plate.<br />
+
       <td width="85" valign="top" style="width:63.8pt;border-top:none;border-left:solid #4BACC6 1.0pt;border-bottom:none;border-right:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.95pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
      - The plate reader made by Molecular  Device then read. <br />
+
       <td width="95" valign="top" style="width:70.9pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.95pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
      - The program does the following: <br />
+
      <td width="94" valign="top" style="width:70.85pt;border-top:none;border-left:solid #4BACC6 1.0pt;border-bottom:none;border-right:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.95pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
      - In Endpoint Reads, following measurements are taken in a time interval of 15 minutes: absorbance (600 nm filter) and fluorescence (485nm and 520nm for GFP). <br />
+
    </tr>
-
      - The results then transfer to excel sheet and interpret.</p>
+
    <tr style="height:12.05pt;">
-
    <p><span class="subtitle"><strong>Immobilization</strong><br />
+
       <td width="73" valign="top" style="width:55.05pt;border:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:12.05pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">SpeI/&mu;L</span></strong></p></td>
-
      <strong>Prepare Sodiumcellu-losesulfate (NaCS)</strong></span><br />
+
       <td width="76" valign="top" style="width:2.0cm;border-top:solid #4BACC6 1.0pt;border-left:none;border-bottom:solid #4BACC6 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:12.05pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
      - Deepfreeze H2SO4  and absolute alcohol at -20℃ for at least 2 hours;<br />
+
       <td width="76" valign="top" style="width:2.0cm;border:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:12.05pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">5.0</span></strong></p></td>
-
      - Prepare a sulphuric acid and  ethanol solution at the proportion of 1.51:1(120mL H2SO4 and 80mL alcohol), maintaining  it at -18℃ for at least 2 hours;<br />
+
       <td width="85" valign="top" style="width:63.75pt;border-top:solid #4BACC6 1.0pt;border-left:none;border-bottom:solid #4BACC6 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:12.05pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
      - Put the sulphuric acid and  ethanol solution and 500mL industrial alcohol in an ice  box, maintaining them at 0℃ for at least 1 hour;<br />
+
      <td width="85" valign="top" style="width:63.8pt;border:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:12.05pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">5.0</span></strong></p></td>
-
       - Immerse 4g dry absorbent cotton  in the solution in ice-bath for 66 min. Then squeezed out the solution and  rinsed the reacted linters with 0℃  industrial alcohol in draught  cupboard;<br />
+
       <td width="95" valign="top" style="width:70.9pt;border-top:solid #4BACC6 1.0pt;border-left:none;border-bottom:solid #4BACC6 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:12.05pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
      - Squeeze out the alcohol, then put  the linters in 400mL deionized water and regulated pH to about 3. Stir and  dissolve it for 10 min, then filtrate it;<br />
+
      <td width="94" valign="top" style="width:70.85pt;border:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:12.05pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
       - Collected the filtrate and  regulated pH to 9.3 accurately;<br />
+
    </tr>
-
       - Add industrial alcohol gradually  to the solution until there appear the largest volume of white retiary floccule  on the top of the solution;<br />
+
    <tr style="height:10.15pt;">
-
       - Centrifuge the floccule for 5 min at 5,000rpm and collect it;<br />
+
       <td width="73" valign="top" style="width:55.05pt;border-top:none;border-left:solid #4BACC6 1.0pt;border-bottom:none;border-right:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:10.15pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">EcoRI/&mu;L</span></strong></p></td>
-
       - 65℃ drying for at least 24 hours  until it is completely dry, then collect the final production.</p>
+
       <td width="76" valign="top" style="width:2.0cm;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:10.15pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">5.0</span></strong></p></td>
-
     <p class="subtitle"><strong>Prepare microcapsules</strong></p>
+
       <td width="76" valign="top" style="width:2.0cm;border-top:none;border-left:solid #4BACC6 1.0pt;border-bottom:none;border-right:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:10.15pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">5.0</span></strong></p></td>
-
    <p>- Centrifuge 10mL bacteria sample for  3 min at 6000rpm and collect the deposit;<br />
+
       <td width="85" valign="top" style="width:63.75pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:10.15pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
       - Add 10mL NaCS solution and mix  it throughly with the cells;<br />
+
      <td width="85" valign="top" style="width:63.8pt;border-top:none;border-left:solid #4BACC6 1.0pt;border-bottom:none;border-right:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:10.15pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">&mdash;</span></strong></p></td>
-
      - Put a 6% PDMDAAC solution on a magnetic  stirrer and stir it at a certain speed, maintaining a small eddy in the center  of liquid surface;<br />
+
      <td width="95" valign="top" style="width:70.9pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:10.15pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">1.0</span></strong></p></td>
-
       - Drop the mixture into the fringe  of the eddy by a 1mL injector until it form an spheroidic membrane. It takes 10 min to react completely and form microcapsules;<br />
+
      <td width="94" valign="top" style="width:70.85pt;border-top:none;border-left:solid #4BACC6 1.0pt;border-bottom:none;border-right:solid #4BACC6 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:10.15pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">2.0</span></strong></p></td>
-
       - Tip all microcapsules to a  strainer and rinse it with sterile water. Then transfer all microcapsules&nbsp; into&nbsp; LB medium with Ampicillin, 37℃ shaker incubate at 100rpm.</p>
+
    </tr>
-
     <p>&nbsp;</p>
+
    <tr style="height:14.65pt;">
-
  </div>
+
       <td width="73" valign="top" style="width:55.05pt;border:solid #4BACC6 1.0pt;border-top:double #4BACC6 2.25pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.65pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">Total/&mu;L</span></strong></p></td>
-
  <p>&nbsp;</p>
+
      <td width="76" valign="top" style="width:2.0cm;border-top:double #4BACC6 2.25pt;border-left:none;border-bottom:solid #4BACC6 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:14.65pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">100</span></strong></p></td>
 +
      <td width="76" valign="top" style="width:2.0cm;border:solid #4BACC6 1.0pt;border-top:double #4BACC6 2.25pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.65pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">100</span></strong></p></td>
 +
      <td width="85" valign="top" style="width:63.75pt;border-top:double #4BACC6 2.25pt;border-left:none;border-bottom:solid #4BACC6 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:14.65pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">100</span></strong></p></td>
 +
       <td width="85" valign="top" style="width:63.8pt;border:solid #4BACC6 1.0pt;border-top:double #4BACC6 2.25pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.65pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">100</span></strong></p></td>
 +
      <td width="95" valign="top" style="width:70.9pt;border-top:double #4BACC6 2.25pt;border-left:none;border-bottom:solid #4BACC6 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:14.65pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">20</span></strong></p></td>
 +
       <td width="94" valign="top" style="width:70.85pt;border:solid #4BACC6 1.0pt;border-top:double #4BACC6 2.25pt;padding:0cm 5.4pt 0cm 5.4pt;height:14.65pt;"><p class="MsoNormal" align="center" style="text-align:center;"><strong><span style="font-family:'Times New Roman','serif'; font-size:8.0pt; color:black; ">20</span></strong></p></td>
 +
    </tr>
 +
  </table>
 +
</div>
 +
<p class="MsoNormal">&nbsp;</p>
 +
<p class="MsoNormal" align="left" style="margin-left:21.1pt;text-align:left;text-indent:-.2pt;"><span style="color:black; "><img src="image001_0003.jpg" alt="" width="900" height="1" border="0"></span><span style="font-family:'Times New Roman','serif'; color:black; ">- System1</span><span style="font-family:宋体; color:black; ">、</span><span style="font-family:'Times New Roman','serif'; color:black; ">2</span><span style="font-family:宋体; color:black; ">、</span><span style="font-family:'Times New Roman','serif'; color:black; ">3 and 4 are used for  Standard BioBrick Assembly .</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:28.25pt;text-align:left;text-indent:-7.35pt;"><span style="font-family:'Times New Roman','serif'; color:black; ">- System 5 and 6 are used for Restriction analysis. Digestion of  sample: at least 500 ng DNA / 10 &mu;L volume. Digest for 4 h at 37 &deg;C, afterwards inactivated by adding 10x loading buffer and standing for 10 min at room  temperature.</span><span style="font-family:'Times New Roman','serif'; color:black; "> </span></p>
 +
<p class="MsoNormal"><span style="font-family:'Times New Roman','serif'; color:black; ">&nbsp;</span></p>
 +
<h2><a name="#_Toc07" class="subsubtitle">Standard BioBrick Assembly</a></h2>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; color:black; ">- Digestion of insert: 2 &mu;g~5 &mu;g DNA / 100 &mu;L volume, 10x H buffer, EcoRI, SpeI. Digestion and inactivation. Clean up the  insert via gel electrophoresis. When cutting the insert out of the gel, try  avoiding staining or exposure to ultraviolet light of the insert. </span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; color:black; ">- Digestion of vector: 2 &mu;g~5 &mu;g DNA / 100 &mu;L volume, 10 x M  buffer, EcoRI, XbaI. Digestion and inactivation. Clean up the insert via gel  electrophoresis. When cutting the insert out of the gel, try to avoid staining or exposure to ultraviolet light of the insert.</span></p>
 +
<p class="MsoNormal"><span style="font-family:'Times New Roman','serif'; color:black; ">&nbsp;</span></p>
 +
<h2><a name="#_Toc08"><span class="subsubtitle">Suffix Insertion</span> </a></h2>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.05pt;"><span style="font-family:'Times New Roman','serif'; color:black; ">- Digestion of insert: 2 &mu;g~5 &mu;g DNA / 100&mu;L volume, 10x M buffer, XbaI, PstI. Digestion and inactivation. Clean up the insert.</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.05pt;"><span style="font-family:'Times New Roman','serif'; color:black; ">- Digestion of vector : 2 &mu;g~5 &mu;g DNA / 100&mu;L volume, 10x H buffer, SpeI, PstI. Digestion and inactivation. Clean up the vector.</span></p>
 +
<p class="MsoNormal"><span style="font-family:'Times New Roman','serif'; color:black; ">&nbsp;</span></p>
 +
<h2><a name="#_Toc09"><span class="subsubtitle">Ligation</span></a> </h2>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.05pt;"><span style="font-family:'Times New Roman','serif'; color:black; ">- After digestion and clean-up, the next step is ligation. ligation at 16<span style="font-family:宋体; ">℃</span> for 4h or at 4<span style="font-family:宋体; ">℃</span> for 16h. Table 2 is the system of ligation. </span></p>
 +
<p class="MsoNormal" align="center" style="text-align:center;"><span style="font-family:'Times New Roman','serif'; color:black; ">Table  2&nbsp; Ligation system</span></p>
 +
<div align="center">
 +
  <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" style="border-collapse:collapse;border:none;">
 +
    <tr style="page-break-inside:avoid;">
 +
      <td width="284" valign="top" style="width:213.05pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal" align="center" style="text-align:center;text-indent:21.1pt;"><strong><span style="font-family:'Times New Roman','serif'; font-size:10.0pt; color:black; ">Components</span></strong><span style="font-family:'Times New Roman','serif'; color:black; "> </span></p></td>
 +
       <td width="284" valign="top" style="width:213.05pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-family:'Times New Roman','serif'; font-size:10.0pt; color:black; ">Volume/</span><span style="font-family:'Times New Roman','serif'; font-size:10.0pt; color:black; ">&mu;L</span><span style="font-family:'Times New Roman','serif'; color:black; "> </span></p></td>
 +
    </tr>
 +
    <tr style="page-break-inside:avoid;">
 +
       <td width="284" valign="top" style="width:213.05pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-family:'Times New Roman','serif'; font-size:10.0pt; color:black; ">Digested vector</span><span style="font-family:'Times New Roman','serif'; color:black; "> </span></p></td>
 +
       <td width="284" valign="top" style="width:213.05pt;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" align="center" style="text-align:center;"><span style="font-family:'Times New Roman','serif'; font-size:10.0pt; color:black; ">7</span><span style="font-family:'Times New Roman','serif'; color:black; "> </span></p></td>
 +
    </tr>
 +
    <tr style="page-break-inside:avoid;">
 +
       <td width="284" valign="top" style="width:213.05pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-family:'Times New Roman','serif'; font-size:10.0pt; color:black; ">Digested insert</span><span style="font-family:'Times New Roman','serif'; color:black; "> </span></p></td>
 +
       <td width="284" valign="top" style="width:213.05pt;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" align="center" style="text-align:center;"><span style="font-family:'Times New Roman','serif'; font-size:10.0pt; color:black; ">1</span><span style="font-family:'Times New Roman','serif'; color:black; "> </span></p></td>
 +
     </tr>
 +
    <tr style="page-break-inside:avoid;">
 +
      <td width="284" valign="top" style="width:213.05pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-family:'Times New Roman','serif'; font-size:10.0pt; color:black; ">10<strong>&times; T<sub>4</sub> ligation buffer</strong></span><span style="font-family:'Times New Roman','serif'; color:black; "> </span></p></td>
 +
       <td width="284" valign="top" style="width:213.05pt;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" align="center" style="text-align:center;"><span style="font-family:'Times New Roman','serif'; font-size:10.0pt; color:black; ">1</span><span style="font-family:'Times New Roman','serif'; color:black; "> </span></p></td>
 +
    </tr>
 +
    <tr style="page-break-inside:avoid;">
 +
       <td width="284" valign="top" style="width:213.05pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-family:'Times New Roman','serif'; font-size:10.0pt; color:black; ">T<sub>4</sub> ligase</span><span style="font-family:'Times New Roman','serif'; color:black; "> </span></p></td>
 +
       <td width="284" valign="top" style="width:213.05pt;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" align="center" style="text-align:center;"><span style="font-family:'Times New Roman','serif'; font-size:10.0pt; color:black; ">1</span><span style="font-family:'Times New Roman','serif'; color:black; "> </span></p></td>
 +
     </tr>
 +
    <tr style="page-break-inside:avoid;">
 +
      <td width="284" valign="top" style="width:213.05pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal" align="center" style="text-align:center;"><span style="font-family:'Times New Roman','serif'; font-size:10.0pt; color:black; ">Total</span><span style="font-family:'Times New Roman','serif'; color:black; "> </span></p></td>
 +
      <td width="284" valign="top" style="width:213.05pt;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" align="center" style="text-align:center;"><span style="font-family:'Times New Roman','serif'; font-size:10.0pt; color:black; ">10</span><span style="font-family:'Times New Roman','serif'; color:black; "> </span></p></td>
 +
    </tr>
 +
  </table>
</div>
</div>
 +
<p class="MsoNormal"><span style="font-family:'Times New Roman','serif'; color:red; ">&nbsp;</span></p>
 +
<p class="MsoNormal"><span style="font-family:'Times New Roman','serif'; color:red; ">&nbsp;</span></p>
 +
<h2><a name="#_Toc10" class="subsubtitle">Restriction  analysis</a> <span style="font-family:'Times New Roman','serif'; font-size:11.5pt; color:black; font-weight:normal; "> </span></h2>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.05pt;"><span style="font-family:'Times New Roman','serif'; color:black; ">- Pick one colony with a sterile tip and cultivation in 20mL LB  for overnight at 37 <span style="font-family:宋体; ">℃</span></span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.05pt;"><span style="font-family:'Times New Roman','serif'; color:black; ">- Isolation of Plasmid</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.05pt;"><span style="font-family:'Times New Roman','serif'; color:black; ">- Digest BioBrick</span><span style="font-family:宋体; color:black; ">,</span><span style="font-family:'Times New Roman','serif'; color:black; ">the system of Restriction analysis refer to table1</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.05pt;"><span style="font-family:'Times New Roman','serif'; color:black; ">- Gel electrophoresis</span><span style="font-family:宋体; color:black; ">:</span><span style="font-family:'Times New Roman','serif'; color:black; ">add 2.2 &mu;L loading buffer to digestion  mixture. An agarose concentration is 1 %.</span></p>
 +
<p class="MsoNormal" style="margin-left:28.25pt;text-indent:-6.95pt;"><span style="font-family:'Times New Roman','serif'; ">&nbsp;</span></p>
 +
<h2><a name="#_Toc11" class="subsubtitle">Gel  Extraction</a> <span style="font-family:'Times New Roman','serif'; font-size:11.5pt; font-weight:normal; "> </span></h2>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- </span><span style="font-family:'Times New Roman','serif'; ">Weigh a 1.5mL centrifuge tube  for each DNA fragment to be isolated and record the weight.</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- </span><span style="font-family:'Times New Roman','serif'; ">Excise gel slice containing the  DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as  possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5  Ml tube and weigh. Record the weight of the gel slice.</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- </span><span style="font-family:'Times New Roman','serif'; ">Add Bing Buffer BD at a ratio  of 100&mu;L of solution per 100mg of agarose gel slices.</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- </span><span style="font-family:'Times New Roman','serif'; ">Incubate the gel mixture at 55-65℃ for 7-10 min or until the gel slice  is completely dissolved. Mix the tube by inversion every few minutes to  facilitate the melting process. Ensure that the gel is completely dissolved.</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- </span><span style="font-family:'Times New Roman','serif'; ">After the dissolved gel mixture  cool down, transfer it to the Spin Columns assembly and incubate for 2 minute  at room temperature.</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- </span><span style="font-family:'Times New Roman','serif'; ">Centrifuge the Spin Columns  assembly in a microcentrifuge at 12,000 rpm for 1 minute, then discard the flow-through.</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- </span><span style="font-family:'Times New Roman','serif'; ">Wash the columns by adding 500 &mu;L  of Wash Buffer PE to the Columns. Centrifuge the columns assembly for 1 minute  at 12,000 rpm, then discard the flow-through.</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- </span><span style="font-family:'Times New Roman','serif'; ">Repeat step 7 again.</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- </span><span style="font-family:'Times New Roman','serif'; ">Centrifuge the Columns for an  additional 3 min to completely remove residual wash buffer.</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- </span><span style="font-family:'Times New Roman','serif'; ">Empty the Collection Tube and  recentrifuge the column assembly for 1 minute with the microcentrifuge lid open  (or off) to allow evaporation of any residual ethanol.</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- </span><span style="font-family:'Times New Roman','serif'; ">Place the spin column in a  clean 1.5 mL microcentrifuge tube, and pipet 20 &mu;L deionized water (pH is  8.0-8.5 and prewarm to 60<span style="font-family:宋体; ">℃</span>)directly to the center of the column  without touching the membrane. Incubate at room temperature for 2 min.</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><a></a><a><span style="font-family:'Times New Roman','serif'; ">-</span></a><span style="font-family:'Times New Roman','serif'; "> </span><span style="font-family:'Times New Roman','serif'; ">Centrifuge for 1 minute at  12,000 rpm. Discard the columns and store the microcentrifuge tube containing  the eluted DNA at </span><span style="font-family:宋体; ">&ndash;</span><span style="font-family:'Times New Roman','serif'; ">20</span><span style="font-family:宋体; ">℃</span><span style="font-family:'Times New Roman','serif'; ">.</span></p>
 +
<p class="MsoNormal" style="margin-left:1.0cm;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">&nbsp;</span></p>
 +
<h1><a name="#_Toc12" class="subtitle">2.Characterization</a> <span style="font-family:'Times New Roman','serif'; font-size:12.0pt; font-weight:normal; "> </span></h1>
 +
<h2><a name="#_Toc13" class="subsubtitle">Fluorescence  Measurements</a> <span style="font-family:'Times New Roman','serif'; font-size:11.5pt; font-weight:normal; "> </span></h2>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- The samples to be  tested are cultured from plates in 20 ml of the Basal Minimal Medium with  appropriate antibiotics and incubated overnight at 37 &deg;C at 200 rpm. </span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- The culture is  checked for OD600 next day and then subculture by the same medium with  antibiotics at 37 &deg;C shaking for 2 hours.&nbsp; </span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- Add corresponding  inducer at concentration gradients into the above-mentioned culture and keep on  incubating. During the time incubating, every 15 min, take 1 mL bacteria  liquid, then centrifuge the cells( 6000 rpm, 10 min ) and resuspend them in 1  mL PBS. At last, pipette to a 96 well plate.</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- The plate reader made  by Molecular Device then read. </span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- The program does the  following: </span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:49.55pt;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- In Endpoint Reads,  following measurements are taken in a time interval of 15 minutes: absorbance (600  nm filter) and fluorescence (485nm and 520nm for GFP). </span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- T</span><span style="font-family:'Times New Roman','serif'; ">he results then  transfer to excel sheet and interpret.</span></p>
 +
<p class="MsoNormal" style="margin-left:1.0cm;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">&nbsp;</span></p>
 +
<h1><a name="#_Toc14" class="subtitle">3.Immobilization</a> <span style="font-family:'Times New Roman','serif'; font-size:12.0pt; font-weight:normal; "> </span></h1>
 +
<h2><a name="#_Toc15" class="subsubtitle">Prepare  Sodiumcellu-losesulfate (NaCS)</a> <span style="font-family:'Times New Roman','serif'; font-size:11.5pt; font-weight:normal; "> </span></h2>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- Deepfreeze H2SO4 and absolute alcohol at -20</span><span style="font-family:宋体; ">℃</span><span style="font-family:'Times New Roman','serif'; "> for at least 2 hours;</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- Prepare a sulphuric  acid and ethanol solution at the proportion of 1.51:1(120mL H2SO4 and 80mL  alcohol), maintaining it at -18</span><span style="font-family:宋体; ">℃</span><span style="font-family:'Times New Roman','serif'; "> for at least 2 hours;</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- Put the sulphuric  acid and ethanol solution and 500mL industrial alcohol  in an ice box, maintaining them at 0</span><span style="font-family:宋体; ">℃</span><span style="font-family:'Times New Roman','serif'; "> for at least 1 hour;</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- Immerse 4g dry  absorbent cotton in the solution in ice-bath for 66 min. Then squeezed out the  solution and rinsed the reacted linters with 0</span><span style="font-family:宋体; ">℃</span><span style="font-family:'Times New Roman','serif'; "> industrial alcohol in draught  cupboard;</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- Squeeze out the  alcohol, then put the linters in 400mL deionized water and regulated pH to  about 3. Stir and dissolve it for 10 min, then filtrate it;</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- Collected the filtrate  and regulated pH to 9.3 accurately;</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- Add industrial  alcohol gradually to the solution until there appear the largest volume of  white retiary floccule on the top of the solution;</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- Centrifuge the  floccule for 5 min at 5,000rpm and collect it;</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- </span><span style="font-family:'Times New Roman','serif'; ">65</span><span style="font-family:宋体; ">℃</span><span style="font-family:'Times New Roman','serif'; "> drying for at least 24 hours  until it is completely dry, then collect the final production.</span></p>
 +
<p class="MsoNormal" style="margin-left:1.0cm;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">&nbsp;</span></p>
 +
<h2><a name="#_Toc16" class="subsubtitle">Prepare  microcapsules</a> <span style="font-family:'Times New Roman','serif'; font-size:11.5pt; font-weight:normal; "> </span></h2>
 +
<p class="MsoNormal" style="margin-left:1.0cm;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">&nbsp;</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- Centrifuge 10mL bacteria  sample for 3 min at 6000rpm and collect the deposit;</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- Add 10mL NaCS  solution and mix it throughly with the cells;</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- Put a 6% PDMDAAC  solution on a magnetic stirrer and stir it at a certain speed, maintaining a  small eddy in the center of liquid surface;</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- Drop the mixture  into the fringe of the eddy by a 1mL injector until it form an spheroidic  membrane. It takes 10 min to react completely and form microcapsules;</span></p>
 +
<p class="MsoNormal" align="left" style="margin-left:1.0cm;text-align:left;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">- Tip all  microcapsules to a strainer and rinse it with sterile water. Then transfer all  microcapsules&nbsp; into&nbsp; LB medium with Ampicillin, 37</span><span style="font-family:宋体; ">℃</span><span style="font-family:'Times New Roman','serif'; "> shaker incubate at 100rpm.</span></p>
 +
<p class="MsoNormal" style="margin-left:1.0cm;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">&nbsp;</span></p>
 +
<p class="MsoNormal" style="margin-left:1.0cm;text-indent:-7.15pt;"><span style="font-family:'Times New Roman','serif'; ">&nbsp;</span></p>
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Revision as of 13:57, 9 September 2012

XMU-CSS

XMU

safetyindex
Contents[hide] [show]
  • 1 General protocols
  •        1.1 Stock solution
  •        1.2 Preparation of Competent BL21
  •        1.3 Transformation
  •        1.4 Plasmid Purification
  •        1.5 Reaction system of restriction endonuclease
  •        1.6 Standard BioBrick Assembly
  •        1.7 Suffix Insertion
  •        1.8 Ligation
  •        1.9 Restriction analysis
  •        1.10 Gel Extraction
  • 2 Characterization
  •        2.1 Fluorescence Measurements
  •        2.2 Protein electrophoresis
  •               2.2.1 Preparation of Samples
  •               2.2.2 Manufacture Albumen Gel
  •               2.2.3 Electrophoresis
  •               2.2.4 Dyeing (Colloidal Coomassie Brilliant Blue)
  •               2.2.5 Scanning
  • 3 Immobilization
  •        3.1 Prepare Sodiumcellu-losesulfate (NaCS)
  •        3.2 Prepare microcapsules
  •        3.3 Immobilize cells into calcium alginate beads
  •        3.4 Immobilize cells into intra-hollow Ca-alginate capsules
  •        3.5 Immobilize cells into NaCS-PDMDAAC microcapsules
  • XMU-CSS

    Protocols

    1.General protocols

    Stock solution

    50mg/mL Kanamycin

    - 0.5g Kan, 10mL water, filter sterilize with millipore express membrane, freeze in aliquots

    100mg/mL Amp

    - 1g Amp, 10mL water, filter sterilize with millipore express membrane, freeze in aliquots.

    50mmol/L Arabinose

    - 0.1876g Arabinose, 25mL water, filter sterilize with millipore express membrane.

     

    Preparation of Competent BL21

    - Thaw an aliquot of cells (without any plasmid in them) on ice

    - To 50 mL of sterile LB, add 100μL aliquot of the thawed cells: remember, this LB does not have any antibiotic in it, so work as aseptically as possible (i.e. autoclave all solutions and use sterile pipettes).

    - Grow cells in the shaker at 37℃ until they reach an OD = 0.3-0.4 at 600nm (1cm pathlength cell). This usually takes 1.5-2 hours.

    - Ice down the LB with growing cells for 10 min.

    - Aliquot into sterile 1.5mL tubes and Spin down at 6000rpm for 10 minutes at 4℃; discard supernatant and.

    - Ice down sterile 100mM CaCl2 and 100mM MgCl2 solutions during centrifugation.

    - Gently resuspend each pellet in 8mL 0.1M MgCl2 and 2mL 0.1 M CaCl2 and combine into 2 tubes (this should take 1-2 minutes per tube)

    - Centrifuge 6000rpm for 10 minutes and discard supernatant.

    - Resuspend each pellet on ice in 100μL 0.1M of ice cold CaCl2 and combine into one tube

     

    Transformation

    - Add 10μL of DNA. Swirl gently with pipette.

    - Incubate tubes on ice for 20 minutes

    - Heat pulse tubes in 42℃ water bath for 30 seconds.

    - Incubate on ice for 2 minutes

    - Add 790uL of LB broth to each tube and incubate for an hour at 37℃ with shaking.

    - Spread 100uL and 50uL of each culture on an LB agar plate containing the appropriate antibiotics and incubate overnight at 37℃ (spread using beads).

     

    Purity Plasmid Miniprep

    - Centrifuge sample in Eppendorf tube approximately 1.5 mL at a time, draining off supernatant after each spin and adding more cell solution

    - Resuspened the pelleted cells in 250 μL of the Resuspension Solution (Mixture with Solution I and RNasa A). The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.

    - Add 250 μL of the Lysis Solution (Solution II) and mix thoroughly and gently by inverting the tube 5-6 times, letting it stand for 1-2minutes at room temperature until the solution becomes viscous and slightly clear.

    - Add 350 μL of the Neutralization Solution (Solution III) and mix immediately and thoroughly by inverting the tube 5-6 times.

    - Centrifuge for 10 min at 12,000rpm to pellet cell debris.

    - Apply the supernatant to the supplied spin column by decanting. Avoid disturbing or applying the white precipitate.

    - Centrifuge for 1 min at 12,000rpm. Discard flow-through and place the column back into the same collection tube.

    - Add 500 μL of the Wash Buffer PB to the spin column. Centrifuge for 1minute at 12,000rpm and discard flow-through. Place the column back into the same collection tube.

    - Add 500 μL of the Wash Buffer W to the spin column. Centrifuge for 1minute at 12,000rpm and discard the flow-through. Place the column back into the same collection tube.

    - Repeat the step 9 again.

    - Discard flow-through and centrifuge for an additional 3 min to remove residual Wash Solution.

    - Place the spin column in a clean 1.5 mL centrifuge tube, and pipet 20 μL Elution Buffer TE (prewarm to 60) directly to the center of the column without touching the membrane. Let it stand for 2 min at room temperature and centrifuge for 1 min at 12,000rpm.

    - Discard the column and store the purified plasmid DNA at -20°C.

     

    Reaction system of restriction endonuclease

     

    Table 1. The digestion system

     

    Prdfix Insertion

    Suffix Insertion

    Restriction analysis

    Components

    EcoRI-XbaI

    EcoRI-SpeI

    PstI-XbaI

    PstI-SpeI

    Double digestion

    Single digestion

    DNA Sample/μL

    80

    80

    80

    80

    16

    16

    10×M buffer/μL

    10

    10

    10×H buffer/μL

    10

    10

    2.0

    2.0

    PstI/μL

    5.0

    5.0

    1.0

    XbaI/μL

    5.0

    5.0

    SpeI/μL

    5.0

    5.0

    EcoRI/μL

    5.0

    5.0

    1.0

    2.0

    Total/μL

    100

    100

    100

    100

    20

    20

     

    - System123 and 4 are used for Standard BioBrick Assembly .

    - System 5 and 6 are used for Restriction analysis. Digestion of sample: at least 500 ng DNA / 10 μL volume. Digest for 4 h at 37 °C, afterwards inactivated by adding 10x loading buffer and standing for 10 min at room temperature.

     

    Standard BioBrick Assembly

    - Digestion of insert: 2 μg~5 μg DNA / 100 μL volume, 10x H buffer, EcoRI, SpeI. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try avoiding staining or exposure to ultraviolet light of the insert.

    - Digestion of vector: 2 μg~5 μg DNA / 100 μL volume, 10 x M buffer, EcoRI, XbaI. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try to avoid staining or exposure to ultraviolet light of the insert.

     

    Suffix Insertion

    - Digestion of insert: 2 μg~5 μg DNA / 100μL volume, 10x M buffer, XbaI, PstI. Digestion and inactivation. Clean up the insert.

    - Digestion of vector : 2 μg~5 μg DNA / 100μL volume, 10x H buffer, SpeI, PstI. Digestion and inactivation. Clean up the vector.

     

    Ligation

    - After digestion and clean-up, the next step is ligation. ligation at 16 for 4h or at 4 for 16h. Table 2 is the system of ligation.

    Table 2  Ligation system

    Components

    Volume/μL

    Digested vector

    7

    Digested insert

    1

    10× T4 ligation buffer

    1

    T4 ligase

    1

    Total

    10

     

     

    Restriction analysis

    - Pick one colony with a sterile tip and cultivation in 20mL LB for overnight at 37

    - Isolation of Plasmid

    - Digest BioBrickthe system of Restriction analysis refer to table1

    - Gel electrophoresisadd 2.2 μL loading buffer to digestion mixture. An agarose concentration is 1 %.

     

    Gel Extraction

    - Weigh a 1.5mL centrifuge tube for each DNA fragment to be isolated and record the weight.

    - Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 Ml tube and weigh. Record the weight of the gel slice.

    - Add Bing Buffer BD at a ratio of 100μL of solution per 100mg of agarose gel slices.

    - Incubate the gel mixture at 55-65℃ for 7-10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved.

    - After the dissolved gel mixture cool down, transfer it to the Spin Columns assembly and incubate for 2 minute at room temperature.

    - Centrifuge the Spin Columns assembly in a microcentrifuge at 12,000 rpm for 1 minute, then discard the flow-through.

    - Wash the columns by adding 500 μL of Wash Buffer PE to the Columns. Centrifuge the columns assembly for 1 minute at 12,000 rpm, then discard the flow-through.

    - Repeat step 7 again.

    - Centrifuge the Columns for an additional 3 min to completely remove residual wash buffer.

    - Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.

    - Place the spin column in a clean 1.5 mL microcentrifuge tube, and pipet 20 μL deionized water (pH is 8.0-8.5 and prewarm to 60)directly to the center of the column without touching the membrane. Incubate at room temperature for 2 min.

    - Centrifuge for 1 minute at 12,000 rpm. Discard the columns and store the microcentrifuge tube containing the eluted DNA at 20.

     

    2.Characterization

    Fluorescence Measurements

    - The samples to be tested are cultured from plates in 20 ml of the Basal Minimal Medium with appropriate antibiotics and incubated overnight at 37 °C at 200 rpm.

    - The culture is checked for OD600 next day and then subculture by the same medium with antibiotics at 37 °C shaking for 2 hours. 

    - Add corresponding inducer at concentration gradients into the above-mentioned culture and keep on incubating. During the time incubating, every 15 min, take 1 mL bacteria liquid, then centrifuge the cells( 6000 rpm, 10 min ) and resuspend them in 1 mL PBS. At last, pipette to a 96 well plate.

    - The plate reader made by Molecular Device then read.

    - The program does the following:

    - In Endpoint Reads, following measurements are taken in a time interval of 15 minutes: absorbance (600 nm filter) and fluorescence (485nm and 520nm for GFP).

    - The results then transfer to excel sheet and interpret.

     

    3.Immobilization

    Prepare Sodiumcellu-losesulfate (NaCS)

    - Deepfreeze H2SO4 and absolute alcohol at -20 for at least 2 hours;

    - Prepare a sulphuric acid and ethanol solution at the proportion of 1.51:1(120mL H2SO4 and 80mL alcohol), maintaining it at -18 for at least 2 hours;

    - Put the sulphuric acid and ethanol solution and 500mL industrial alcohol in an ice box, maintaining them at 0 for at least 1 hour;

    - Immerse 4g dry absorbent cotton in the solution in ice-bath for 66 min. Then squeezed out the solution and rinsed the reacted linters with 0 industrial alcohol in draught cupboard;

    - Squeeze out the alcohol, then put the linters in 400mL deionized water and regulated pH to about 3. Stir and dissolve it for 10 min, then filtrate it;

    - Collected the filtrate and regulated pH to 9.3 accurately;

    - Add industrial alcohol gradually to the solution until there appear the largest volume of white retiary floccule on the top of the solution;

    - Centrifuge the floccule for 5 min at 5,000rpm and collect it;

    - 65 drying for at least 24 hours until it is completely dry, then collect the final production.

     

    Prepare microcapsules

     

    - Centrifuge 10mL bacteria sample for 3 min at 6000rpm and collect the deposit;

    - Add 10mL NaCS solution and mix it throughly with the cells;

    - Put a 6% PDMDAAC solution on a magnetic stirrer and stir it at a certain speed, maintaining a small eddy in the center of liquid surface;

    - Drop the mixture into the fringe of the eddy by a 1mL injector until it form an spheroidic membrane. It takes 10 min to react completely and form microcapsules;

    - Tip all microcapsules to a strainer and rinse it with sterile water. Then transfer all microcapsules  into  LB medium with Ampicillin, 37 shaker incubate at 100rpm.