Team:XMU-China/protocols

From 2012.igem.org

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<p ><span class="tit">Protocols</span><br />
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  <div id="subpage2">
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  <strong ><span class="subtitle">General  protocols</span></strong><br />
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    <p ><span class="tit">Protocols</span><br />
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  <strong class="subtitle">Stock solution</strong><br />
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      <strong ><span class="subtitle">General  protocols</span></strong><br />
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  50mg/mL  Kanamycin <br />
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      <strong class="subtitle">Stock solution</strong><br />
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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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      50mg/mL  Kanamycin <br />
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  100mg/mL  Amp <br />
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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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  -1g Amp, 10mL  water, filter <a name="OLE_LINK1" id="OLE_LINK1">sterilize</a> with millipore express  membrane, freeze in aliquots. <br />
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      100mg/mL  Amp <br />
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  50mmol/L  Arabinose<br />
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      -1g Amp, 10mL  water, filter <a name="OLE_LINK1" id="OLE_LINK1">sterilize</a> with millipore express  membrane, freeze in aliquots. <br />
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-0.1876g Arabinose, 25mL water, filter sterilize  with millipore express membrane.</p>
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      50mmol/L  Arabinose<br />
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<p><strong class="subtitle">Preparation of  Competent BL21 </strong> <br />
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      -0.1876g Arabinose, 25mL water, filter sterilize  with millipore express membrane.</p>
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  <a name="OLE_LINK10" id="OLE_LINK10"></a><a name="OLE_LINK9" id="OLE_LINK9">-</a> Thaw an aliquot of cells (without any  plasmid in them) on ice <br />
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    <p><strong class="subtitle">Preparation of  Competent BL21 </strong> <br />
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  - To 50 mL of <a name="OLE_LINK6" id="OLE_LINK6"></a><a name="OLE_LINK3" id="OLE_LINK3">sterile</a> 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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      <a name="OLE_LINK10" id="OLE_LINK10"></a><a name="OLE_LINK9" id="OLE_LINK9">-</a> Thaw an aliquot of cells (without any  plasmid in them) on ice <br />
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  - Grow cells in the shaker at 37<a name="OLE_LINK5" id="OLE_LINK5"></a><a name="OLE_LINK4" id="OLE_LINK4">℃</a> 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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      - To 50 mL of <a name="OLE_LINK6" id="OLE_LINK6"></a><a name="OLE_LINK3" id="OLE_LINK3">sterile</a> 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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  - Ice down the  LB with growing cells for 10 min.<br />
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      - Grow cells in the shaker at 37<a name="OLE_LINK5" id="OLE_LINK5"></a><a name="OLE_LINK4" id="OLE_LINK4">℃</a> 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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  - Aliquot into  sterile 1.5mL tubes and Spin down at 6000rpm for 10 minutes at 4℃; discard supernatant and.<br />
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      - Ice down the  LB with growing cells for 10 min.<br />
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  - <a name="OLE_LINK2" id="OLE_LINK2">Ice down sterile</a> 100mM CaCl2 and 100mM MgCl2 solutions during  centrifugation. <br />
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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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  - 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) <br />
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      - <a name="OLE_LINK2" id="OLE_LINK2">Ice down sterile</a> 100mM CaCl2 and 100mM MgCl2 solutions during  centrifugation. <br />
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  - Centrifuge  6000rpm for 10 minutes and discard supernatant. <br />
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      - 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) <br />
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- Resuspend  each pellet on ice in 100&mu;L 0.1M  of ice cold CaCl2 and combine into one tube </p>
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      - Centrifuge  6000rpm for 10 minutes and discard supernatant. <br />
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<p><strong class="subtitle">Transformation </strong> <br />
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      - Resuspend  each pellet on ice in 100&mu;L 0.1M  of ice cold CaCl2 and combine into one tube </p>
-
  - Add 10&mu;L of DNA. Swirl gently with  pipette. <br />
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    <p><strong class="subtitle">Transformation </strong> <br />
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  - Incubate tubes on ice for 20 minutes <br />
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      - Add 10&mu;L of DNA. Swirl gently with  pipette. <br />
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  - Heat pulse tubes in 42℃ water bath for 30 seconds. <br />
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      - Incubate tubes on ice for 20 minutes <br />
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  - Incubate on ice for 2 minutes <br />
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      - Heat pulse tubes in 42℃ water bath for 30 seconds. <br />
-
  - Add 790uL of LB broth to each tube and incubate for an hour at 37℃ with shaking. <br />
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      - Incubate on ice for 2 minutes <br />
-
<a name="OLE_LINK19" id="OLE_LINK19"></a><a name="OLE_LINK14" id="OLE_LINK14"></a><a name="OLE_LINK13" id="OLE_LINK13">- </a>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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      - Add 790uL of LB broth to each tube and incubate for an hour at 37℃ with shaking. <br />
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<p><strong class="subtitle">Purity Plasmid Miniprep</strong> <br />
+
      <a name="OLE_LINK19" id="OLE_LINK19"></a><a name="OLE_LINK14" id="OLE_LINK14"></a><a name="OLE_LINK13" id="OLE_LINK13">- </a>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>
-
  <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><strong class="subtitle">Purity Plasmid Miniprep</strong> <br />
-
  <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 />
+
      <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 />
-
  <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 />
+
      <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 />
-
  <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 />
+
      <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 />
-
  - Centrifuge for 10 min at  12,000rpm to pellet cell debris.<br />
+
      <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 />
-
  - Apply the supernatant to the  supplied spin column by decanting. Avoid disturbing or applying the white  precipitate.<br />
+
      - Centrifuge for 10 min at  12,000rpm to pellet cell debris.<br />
-
  - Centrifuge for 1 min at  12,000rpm. Discard  flow-through and place the column back into the same collection tube.<br />
+
      - Apply the supernatant to the  supplied spin column by decanting. Avoid disturbing or applying the white  precipitate.<br />
-
  - 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 />
+
      - Centrifuge for 1 min at  12,000rpm. Discard  flow-through and place the column back into the same collection tube.<br />
-
  - 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 />
+
      - 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 />
-
  - Repeat the step 9 again.<br />
+
      - 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 />
-
  <a name="OLE_LINK18" id="OLE_LINK18"></a><a name="OLE_LINK17" id="OLE_LINK17">-</a> Discard flow-through  and centrifuge for an additional 3 min to remove residual Wash Solution. <br />
+
      - Repeat the step 9 again.<br />
-
  - 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 />
+
      <a name="OLE_LINK18" id="OLE_LINK18"></a><a name="OLE_LINK17" id="OLE_LINK17">-</a> Discard flow-through  and centrifuge for an additional 3 min to remove residual Wash Solution. <br />
-
<a name="OLE_LINK16" id="OLE_LINK16"></a><a name="OLE_LINK15" id="OLE_LINK15">- </a>Discard the column and  store the purified plasmid DNA at -20&deg;C.</p>
+
      - 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 />
-
<p><a name="OLE_LINK31" id="OLE_LINK31"></a><a name="OLE_LINK30" class="subtitle" id="OLE_LINK30"><strong>Reaction  system of restriction endonuclease</strong></a></p>
+
      <a name="OLE_LINK16" id="OLE_LINK16"></a><a name="OLE_LINK15" id="OLE_LINK15">- </a>Discard the column and  store the purified plasmid DNA at -20&deg;C.</p>
-
<p align="left"><img width="553" height="297" src="file:///D|/web/webstudio/image002.jpg" /><img width="900" height="1" src="file:///D|/web/webstudio/image003.jpg" />- System1、2、3 and 4 are used for Standard BioBrick Assembly .<br />
+
    <p><a name="OLE_LINK31" id="OLE_LINK31"></a><a name="OLE_LINK30" class="subtitle" id="OLE_LINK30"><strong>Reaction  system of restriction endonuclease</strong></a></p>
-
  - 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>
+
    <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="file:///D|/web/webstudio/image003.jpg" />- System1、2、3 and 4 are used for Standard BioBrick Assembly .<br />
-
<p><strong class="subtitle">Standard BioBrick Assembly</strong><br />
+
      - 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>
-
  - 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 />
+
    <p><strong class="subtitle">Standard BioBrick Assembly</strong><br />
-
- 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>
+
      - 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 />
-
<p><strong class="subtitle">Suffix Insertion </strong><br />
+
      - 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>
-
  - 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 />
+
    <p><strong class="subtitle">Suffix Insertion </strong><br />
-
- 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>
+
      - 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 />
-
<p><strong class="subtitle">Ligation </strong><br />
+
      - 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>
-
  - 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 />
+
    <p><strong class="subtitle">Ligation </strong><br />
-
Table 2&nbsp; Ligation system</p>
+
      - 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 />
-
<div align="center">
+
      Table 2&nbsp; Ligation system</p>
-
  <table border="1" cellspacing="0" cellpadding="0">
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    <div align="center">
-
    <tr>
+
      <table border="1" cellspacing="0" cellpadding="0">
-
      <td width="284" valign="top"><br />
+
        <tr>
-
        <strong>Components</strong></td>
+
          <td width="284" valign="top"><br />
-
      <td width="284" valign="top"><p align="center">Volume/&micro;L </p></td>
+
            <strong>Components</strong></td>
-
    </tr>
+
          <td width="284" valign="top"><p align="center">Volume/&micro;L </p></td>
-
    <tr>
+
        </tr>
-
      <td width="284" valign="top"><p align="center">Digested vector </p></td>
+
        <tr>
-
      <td width="284" valign="top"><p align="center">7 </p></td>
+
          <td width="284" valign="top"><p align="center">Digested vector </p></td>
-
    </tr>
+
          <td width="284" valign="top"><p align="center">7 </p></td>
-
    <tr>
+
        </tr>
-
      <td width="284" valign="top"><p align="center">Digested insert </p></td>
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        <tr>
-
      <td width="284" valign="top"><p align="center">1 </p></td>
+
          <td width="284" valign="top"><p align="center">Digested insert </p></td>
-
    </tr>
+
          <td width="284" valign="top"><p align="center">1 </p></td>
-
    <tr>
+
        </tr>
-
      <td width="284" valign="top"><p align="center">10<strong>&times; T4 ligation buffer</strong></p></td>
+
        <tr>
-
      <td width="284" valign="top"><p align="center">1 </p></td>
+
          <td width="284" valign="top"><p align="center">10<strong>&times; T4 ligation buffer</strong></p></td>
-
    </tr>
+
          <td width="284" valign="top"><p align="center">1 </p></td>
-
    <tr>
+
        </tr>
-
      <td width="284" valign="top"><p align="center">T4 ligase </p></td>
+
        <tr>
-
      <td width="284" valign="top"><p align="center">1 </p></td>
+
          <td width="284" valign="top"><p align="center">T4 ligase </p></td>
-
    </tr>
+
          <td width="284" valign="top"><p align="center">1 </p></td>
-
    <tr>
+
        </tr>
-
      <td width="284" valign="top"><p align="center">Total </p></td>
+
        <tr>
-
      <td width="284" valign="top"><p align="center">10 </p></td>
+
          <td width="284" valign="top"><p align="center">Total </p></td>
-
    </tr>
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          <td width="284" valign="top"><p align="center">10 </p></td>
-
  </table>
+
        </tr>
 +
      </table>
 +
    </div>
 +
    <p>&nbsp;</p>
 +
    <p><strong class="subtitle">Restriction analysis</strong><br />
 +
      - Pick one colony with a sterile tip and cultivation in 20mL LB for  overnight at 37 ℃<br />
 +
      - Isolation of Plasmid<br />
 +
      - Digest BioBrick,the system of  Restriction analysis refer to table1<br />
 +
      - Gel electrophoresis:add 2.2 &micro;L loading  buffer to digestion mixture. An agarose concentration is 1 %.</p>
 +
    <p><strong class="subtitle">Gel Extraction</strong><br />
 +
      - Weigh a 1.5mL centrifuge tube  for each DNA fragment to be isolated and record the weight.<br />
 +
      - 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 />
 +
      - Add Bing Buffer BD at a ratio  of 100&mu;L of solution per 100mg of agarose gel slices.<br />
 +
      - &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 />
 +
      - &nbsp;After the dissolved gel mixture cool  down, transfer it to the Spin Columns assembly and incubate for 2 minute at  room temperature.<br />
 +
      - Centrifuge the Spin Columns  assembly in a microcentrifuge at 12,000 rpm for 1 minute, then discard the  flow-through.<br />
 +
      - &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 />
 +
      - &nbsp;Repeat step 7 again.<br />
 +
      - Centrifuge the Columns for an  additional 3 min to completely remove residual wash buffer.<br />
 +
      - &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 />
 +
      - 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 />
 +
      <a name="OLE_LINK23" id="OLE_LINK23"></a><a name="OLE_LINK22" id="OLE_LINK22">-</a> Centrifuge for 1 minute at  12,000 rpm. Discard the columns and store the microcentrifuge tube containing  the eluted DNA at &ndash;20℃.</p>
 +
    <p><span class="subtitle"><strong>Characterization</strong><br />
 +
      <strong>Fluorescence Measurements</strong></span><br />
 +
      -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 />
 +
      -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 />
 +
      -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 />
 +
      -The plate reader made by Molecular  Device then read. <br />
 +
      -The program does the following: <br />
 +
      -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 />
 +
      <a name="OLE_LINK27" id="OLE_LINK27"></a><a name="OLE_LINK26" id="OLE_LINK26">-</a>The results then  transfer to excel sheet and interpret.</p>
 +
    <p><span class="subtitle"><strong>Immobilization</strong><br />
 +
      <strong>Prepare Sodiumcellu-losesulfate  (NaCS)</strong></span><br />
 +
      -Deepfreeze <a name="OLE_LINK7" id="OLE_LINK7">H2SO4  and absolute alcohol</a> at -20℃ for at least 2 hours;<br />
 +
      -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 />
 +
      -Put the sulphuric acid and  ethanol solution and 500mL <a name="OLE_LINK8" id="OLE_LINK8">industrial</a> alcohol in an ice  box, maintaining them at 0℃ for at least 1 hour;<br />
 +
      -Immerse 4g dry absorbent cotton  in the solution in ice-bath for 66 min. Then squeezed out the solution and  rinsed the reacted <a name="OLE_LINK12" id="OLE_LINK12">linters</a> with 0℃  industrial <a name="OLE_LINK11" id="OLE_LINK11">alcohol</a> in draught  cupboard;<br />
 +
      -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 />
 +
      -Collected the filtrate and  regulated pH to 9.3 accurately;<br />
 +
      -Add industrial alcohol gradually  to the solution until there appear the largest volume of white retiary floccule  on the top of the solution;<br />
 +
      -Centrifuge the floccule for 5  min at 5,000rpm and collect it;<br />
 +
      <a name="OLE_LINK29" id="OLE_LINK29"></a><a name="OLE_LINK28" id="OLE_LINK28">-</a>65℃ drying for at least 24 hours  until it is completely dry, then collect the final production.</p>
 +
    <p class="subtitle"><strong>Prepare microcapsules</strong></p>
 +
    <p>-Centrifuge 10mL bacteria sample for  3 min at 6000rpm and collect the deposit;<br />
 +
      -Add 10mL NaCS solution and mix  it throughly with the cells;<br />
 +
      -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 />
 +
      -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 />
 +
      -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>
 +
    <p>&nbsp;</p>
 +
  </div>
 +
  <p align="left">&nbsp;</p>
</div>
</div>
-
<p>&nbsp;</p>
 
-
<p><strong class="subtitle">Restriction analysis</strong><br />
 
-
  - Pick one colony with a sterile tip and cultivation in 20mL LB for  overnight at 37 ℃<br />
 
-
  - Isolation of Plasmid<br />
 
-
  - Digest BioBrick,the system of  Restriction analysis refer to table1<br />
 
-
- Gel electrophoresis:add 2.2 &micro;L loading  buffer to digestion mixture. An agarose concentration is 1 %.</p>
 
-
<p><strong class="subtitle">Gel Extraction</strong><br />
 
-
  - Weigh a 1.5mL centrifuge tube  for each DNA fragment to be isolated and record the weight.<br />
 
-
  - 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 />
 
-
  - Add Bing Buffer BD at a ratio  of 100&mu;L of solution per 100mg of agarose gel slices.<br />
 
-
  - &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 />
 
-
  - &nbsp;After the dissolved gel mixture cool  down, transfer it to the Spin Columns assembly and incubate for 2 minute at  room temperature.<br />
 
-
  - Centrifuge the Spin Columns  assembly in a microcentrifuge at 12,000 rpm for 1 minute, then discard the  flow-through.<br />
 
-
  - &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 />
 
-
  - &nbsp;Repeat step 7 again.<br />
 
-
  - Centrifuge the Columns for an  additional 3 min to completely remove residual wash buffer.<br />
 
-
  - &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 />
 
-
  - 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 />
 
-
<a name="OLE_LINK23" id="OLE_LINK23"></a><a name="OLE_LINK22" id="OLE_LINK22">-</a> Centrifuge for 1 minute at  12,000 rpm. Discard the columns and store the microcentrifuge tube containing  the eluted DNA at &ndash;20℃.</p>
 
-
<p><span class="subtitle"><strong>Characterization</strong><br />
 
-
  <strong>Fluorescence Measurements</strong></span><br />
 
-
  -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 />
 
-
  -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 />
 
-
  -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 />
 
-
  -The plate reader made by Molecular  Device then read. <br />
 
-
  -The program does the following: <br />
 
-
  -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 />
 
-
<a name="OLE_LINK27" id="OLE_LINK27"></a><a name="OLE_LINK26" id="OLE_LINK26">-</a>The results then  transfer to excel sheet and interpret.</p>
 
-
<p><span class="subtitle"><strong>Immobilization</strong><br />
 
-
  <strong>Prepare Sodiumcellu-losesulfate  (NaCS)</strong></span><br />
 
-
  -Deepfreeze <a name="OLE_LINK7" id="OLE_LINK7">H2SO4  and absolute alcohol</a> at -20℃ for at least 2 hours;<br />
 
-
  -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 />
 
-
  -Put the sulphuric acid and  ethanol solution and 500mL <a name="OLE_LINK8" id="OLE_LINK8">industrial</a> alcohol in an ice  box, maintaining them at 0℃ for at least 1 hour;<br />
 
-
  -Immerse 4g dry absorbent cotton  in the solution in ice-bath for 66 min. Then squeezed out the solution and  rinsed the reacted <a name="OLE_LINK12" id="OLE_LINK12">linters</a> with 0℃  industrial <a name="OLE_LINK11" id="OLE_LINK11">alcohol</a> in draught  cupboard;<br />
 
-
  -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 />
 
-
  -Collected the filtrate and  regulated pH to 9.3 accurately;<br />
 
-
  -Add industrial alcohol gradually  to the solution until there appear the largest volume of white retiary floccule  on the top of the solution;<br />
 
-
  -Centrifuge the floccule for 5  min at 5,000rpm and collect it;<br />
 
-
<a name="OLE_LINK29" id="OLE_LINK29"></a><a name="OLE_LINK28" id="OLE_LINK28">-</a>65℃ drying for at least 24 hours  until it is completely dry, then collect the final production.</p>
 
-
<p class="subtitle"><strong>Prepare microcapsules</strong></p>
 
-
<p>-Centrifuge 10mL bacteria sample for  3 min at 6000rpm and collect the deposit;<br />
 
-
  -Add 10mL NaCS solution and mix  it throughly with the cells;<br />
 
-
  -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 />
 
-
  -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 />
 
-
-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>
 
-
<p>&nbsp;</p>
 
-
</div>
 
-
 
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Revision as of 15:40, 7 September 2012

XMU

XMU-Protocols

Protocols
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

- System1、2、3 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 / 100 µL volume, 10x M buffer, XbaI, PstI. Digestion and inactivation. Clean up the insert.
- Digestion of vector : 2 μg~5 μg DNA / 100 µL 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 BioBrick,the system of Restriction analysis refer to table1
- Gel electrophoresis:add 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℃.

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.

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.