Tianjin/FLIP-FLOP/More details

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<ul>
<ul>
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<li><font size="4" color="#663366">E.coli Cultivate</font></li>
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<li><font size="4" color="#663366">E.coli Cultivation</font></li>
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Solid LB culture is used for agar plate. For a typical liquid culture, use 5 ml or 100ml of appropriate medium, and condition is supposed to be 37°C and 220rpm. The amount of time you wait depends on the reason for growing the cells. To miniprep plasmid DNA, an overnight culture is sufficient. To get the transformation colony, wait about 12-16 hours.
+
Solid LB medium is used for agar plate. For a typical cultivation, 5 ml or 100ml of appropriate medium is used, and the appopriate condition for cultivation in shakers is 37°C and 220rpm and the time depends on the reason for growing the cells. To miniprep plasmid DNA, an overnight culture is sufficient. To get the transformation colony, waiting for about 12-16 hours.
<li><font size="4" color="#663366">Plasmid Extraction</font></li>
<li><font size="4" color="#663366">Plasmid Extraction</font></li>
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Using plasmid extracting kit we get the plasmid DNA for two purposes. One is verifying the plasmid using enzyme digesting and the other is purifying the fragment using enzyme digesting and agarose gel electrophoresis.
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Using plasmid extracting kit we get the plasmid DNA for two purposes. One is to verify the plasmid using enzyme digesting and the other is to purify the fragments for ligation using enzyme digesting and gel recycle.
</ul>
</ul>
</div>  <!-- end of part1 -->
</div>  <!-- end of part1 -->
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<ul>
<ul>
<li><font size="4" color="#663366">Verification</font></li>
<li><font size="4" color="#663366">Verification</font></li>
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Colony PCR can be used after a transformation to screen colonies for the desired plasmid.But it always give the incorrect result, so often we use enzyme digesting to verify the desired palsmid.
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Colony PCR can be used to screen colonies containing desired plasmid.But it always brings out the incorrect result, so under most conditions we use enzyme digesting to verify the desired palsmid.
<li><font size="4" color="#663366">Restrication digestion</font></li>
<li><font size="4" color="#663366">Restrication digestion</font></li>
<table width="99%" cellpadding="0" cellspacing="20" style="padding: 10px; background-color: #FFFF99">
<table width="99%" cellpadding="0" cellspacing="20" style="padding: 10px; background-color: #FFFF99">
<td><font size="3" color="#9966CC">10μL digesting Mix</font>
<td><font size="3" color="#9966CC">10μL digesting Mix</font>
<div id="10">
<div id="10">
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<ul>
<ul>
<li>1.0 μL 10X Enzyme buffer </li>
<li>1.0 μL 10X Enzyme buffer </li>
<li>5 μL plasmid DNA</li>
<li>5 μL plasmid DNA</li>
<li>0.5 μL Restriction Enzyme respectively</li>
<li>0.5 μL Restriction Enzyme respectively</li>
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<li>Add ddH<sub>2</sub>O to 10ul volume</li>
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<li>Add ddH<sub>2</sub>O to 10ul </li>
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</ul>
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</ul>
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</div>  <!-- end of 10 --></td>
</div>  <!-- end of 10 --></td>
<td><font size="3" color="#9966CC">50μL digesting Mix</font>
<td><font size="3" color="#9966CC">50μL digesting Mix</font>
<div id="50">
<div id="50">
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<ul>
<ul>
<li>5.0 μL 10X Enzyme buffer </li>
<li>5.0 μL 10X Enzyme buffer </li>
<li>30 μL plasmid DNA</li>
<li>30 μL plasmid DNA</li>
<li>2 μL Restriction Enzyme respectively</li>
<li>2 μL Restriction Enzyme respectively</li>
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<li>Add ddH<sub>2</sub>O to 50 ul volume</li>
+
<li>Add ddH<sub>2</sub>O to 50 ul </li>
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+
</ul>
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</ul>
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</div>  <!-- end of 50 -->
</div>  <!-- end of 50 -->
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</td>
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</td></table>
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</table>
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</ul>
</ul>
</div>  <!-- end of part2 -->
</div>  <!-- end of part2 -->
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</div>  <!-- end of agarose -->
</div>  <!-- end of agarose -->
<li><font size="4" color="#663366">DNA ligation</font></li>
<li><font size="4" color="#663366">DNA ligation</font></li>
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The fragment usually is gotten from gel purification, sometimes from nucleic acid co-depositing.<br>
+
The fragment usually is prepared from gel purification, sometimes from nucleic acid co-depositing.<br>
<font size="3" color="#9966CC">Reagents</font>
<font size="3" color="#9966CC">Reagents</font>
<div id="reagents">
<div id="reagents">
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<font size="3" color="#9966CC">Calculating</font><br>
<font size="3" color="#9966CC">Calculating</font><br>
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The amount of vector and insert fragment is decided by formula given by openwetware.
+
The amount of vector and insert fragment needed for a successful ligation is decided by formula given by openwetware.
   
   
<font size="3" color="#9966CC">Reaction</font><br>
<font size="3" color="#9966CC">Reaction</font><br>
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Ligate reaction is taken by TaKaRa DNA Ligation Kit Ver.2.0. Total volume is 10μL, one half is DNA solution and one half is Solution I. Temperature is 16°C. Then waiting for different time according to different situations.
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Ligate reaction is taken by TaKaRa DNA Ligation Kit Ver.2.0. Total reaction volume is 10μL, one half of which is DNA solution and another half is Solution I containing T4 ligation enzyme. Typical reaction temperature is 16°C. Reaction time varies from 45 minutes to 6 hours depending on the specific condition.
</ul>
</ul>
</div>  <!-- end of part3 -->
</div>  <!-- end of part3 -->
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<ul>
<ul>
<li><font size="4" color="#663366">E.coli Transformation</font></li>
<li><font size="4" color="#663366">E.coli Transformation</font></li>
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Using CaCl<sub>2</sub> solution to make competent cells, and chemically transform foreign DNA to competent cells. If foreign DNA is plasmid, using 2-3 μL to transform 100 μL competent cells. If it is ligation product, transform the total 10 μL jproduct to 100 μL competent cells.
+
Using CaCl<sub>2</sub> solution to make competent cells, and foreign DNA is transformed chemically to competent cells. If foreign DNA is plasmid, using 2-3 μL to transform 100 μL competent cells. If it is ligation product, transform the total 10 μL product to 100 μL competent cells.
        <li><font size="4" color="#663366">Detection</font></li>
        <li><font size="4" color="#663366">Detection</font></li>
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Open the bottle of medium and flame the mouth, measure out 2 ml to fill 10 ml Eppendorf Tube.Centrifugate the tube and pipette in 5 ml of the suspension to another tube for detecting the extracellular AHL.Then the deposit is used to detect GFP using fluorometry. AHL is detected by the detector used in Bio-Diode.
+
Opening the bottle of medium and flame the mouth, bringing 2 ml to 10 ml Eppendorf Tube.Centrifugate the tube and pipette in 5 ml of the suspension to another tube for detecting the extracellular AHL.Then the deposit is used to detect GFP using fluorometer. The concentration of AHL is detected by the detector used in Bio-Diode.
</ul>
</ul>
</div>  <!-- end of part4 -->
</div>  <!-- end of part4 -->

Latest revision as of 03:17, 27 October 2007

Experiment

  • E.coli Cultivation

    • Solid LB medium is used for agar plate. For a typical cultivation, 5 ml or 100ml of appropriate medium is used, and the appopriate condition for cultivation in shakers is 37°C and 220rpm and the time depends on the reason for growing the cells. To miniprep plasmid DNA, an overnight culture is sufficient. To get the transformation colony, waiting for about 12-16 hours.

  • Plasmid Extraction

    • Using plasmid extracting kit we get the plasmid DNA for two purposes. One is to verify the plasmid using enzyme digesting and the other is to purify the fragments for ligation using enzyme digesting and gel recycle.

  • Verification

    • Colony PCR can be used to screen colonies containing desired plasmid.But it always brings out the incorrect result, so under most conditions we use enzyme digesting to verify the desired palsmid.

  • Restrication digestion
    • 10μL digesting Mix
    • 1.0 μL 10X Enzyme buffer
    • 5 μL plasmid DNA
    • 0.5 μL Restriction Enzyme respectively
    • Add ddH2O to 10ul
    		50μL digesting Mix
    • 5.0 μL 10X Enzyme buffer
    • 30 μL plasmid DNA
    • 2 μL Restriction Enzyme respectively
    • Add ddH2O to 50 ul
  • Agarose gel electrophoresis
    • Agarose concentration : 1.0g/100mL
    • Buffers: TAE - better resolution of fragments >4kb
    • The appropriate volume: 30 ml for fragment verifying or 50 ml for fragment purification
    • Voltage: 80-100V
  • DNA ligation

    • The fragment usually is prepared from gel purification, sometimes from nucleic acid co-depositing.
    Reagents

    • T4 DNA ligase
    • 10x T4 DNA Ligase Buffer
    • Deionized, sterile H2O
    • Purified, linearized vector in EB
    • Purified, linearized insert in EB

    Calculating
    The amount of vector and insert fragment needed for a successful ligation is decided by formula given by openwetware.

    Reaction
    Ligate reaction is taken by TaKaRa DNA Ligation Kit Ver.2.0. Total reaction volume is 10μL, one half of which is DNA solution and another half is Solution I containing T4 ligation enzyme. Typical reaction temperature is 16°C. Reaction time varies from 45 minutes to 6 hours depending on the specific condition.

  • E.coli Transformation

    • Using CaCl2 solution to make competent cells, and foreign DNA is transformed chemically to competent cells. If foreign DNA is plasmid, using 2-3 μL to transform 100 μL competent cells. If it is ligation product, transform the total 10 μL product to 100 μL competent cells.

  • Detection

    • Opening the bottle of medium and flame the mouth, bringing 2 ml to 10 ml Eppendorf Tube.Centrifugate the tube and pipette in 5 ml of the suspension to another tube for detecting the extracellular AHL.Then the deposit is used to detect GFP using fluorometer. The concentration of AHL is detected by the detector used in Bio-Diode.

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