Team:Tianjin/Protocol

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(Competent Cell (e.g. E.coli BL 21))
 
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<a href=" https://2013.igem.org/Team:Tianjin/Project/Background "> Background </a>
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<a href=" https://2013.igem.org/Team:Tianjin/Project/Alk-Detector "> Alk-Detector </a>
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<a href=" https://2013.igem.org/Team:Tianjin/Project/Selection "> Selection </a>
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<a href=" https://2013.igem.org/Team:Tianjin/Project/In the Future "> In the Future </a>
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<a href=" https://2013.igem.org/Team:Tianjin/Data "> Biobrick </a>
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    <li class="A-C"><a href="https://2013.igem.org/Team:Tianjin/Attributions"  style="padding:7px 0px 7px 0px;">Attributions<br/>&amp;<br/>Contributions</a>
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<div class="cont">
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        <ul>
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            <li class="hmain" style="margin-top:20px;">
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                <a href="#anchor01">Luria Bertani Medium</a>
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            </li>
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            <li class="hmain">
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                <a href="#anchor02">M9 Medium</a>
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            </li>
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            <li class="hmain">
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                <a href="#anchor03">Extracting Alkanes</a>
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            </li>
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            <li class="hmain">
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                <a href="#anchor04">Ligation</a>
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            </li>
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            <li class="hmain" style="font-size:14px;">
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                <a href="#anchor05">Restriction Enzyme Digestion</a>
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            </li>
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            <li class="hmain">
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                <a href="#anchor06">Competent Cell</a>
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            </li>
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            <li class="hmain">
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                <a href="#anchor07">DNA Agarose Gels</a>
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            </li>
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            <li class="hmain">
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                <a href="#anchor08" style="font-size:14px;">Agarose Gel Electrophoresis</a>
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            </li>
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            <li class="hmain">
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                <a href="#anchor09">PCR Purification</a>
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            </li>
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            <li class="hmain">
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                <a href="#anchor10">Gel Extraction of DNA</a>
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            </li>
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            <li class="hmain">
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                <a href="#anchor11">Error-Prone PCR</a>
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            </li>
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            <li class="hmain">
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                <a href="#anchor12">ColonyPCR</a>
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            </li>
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            <li class="hmain">
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                <a href="#anchor13">Fluor Spectrophotometry</a>
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            </li>
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<div class="box">
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<!--代码开始-->
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<center><span style="font-size:46px;font-family:Arial;margin-top:10px;line-height:100%">Project</span></center>
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<p><br/>
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</p>
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<p>The Glossary: </p>
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<ul><li> The O-Key System -- Any orthogonal system containing a pair of orthogonal ribosome and mRNA
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</li><li> The O-Key -- the orthogonal ribosome, which serves like a key to translate the orthogonal mRNA
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</li><li> The O-Lock -- the orthogonal mRNA, which can only be deciphered by the O-Key.
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<h1> <span>Orthogonal System</span></h1>
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+
-
<a href="#" ><img alt="" src="images/pic001.jpg" width="300" height="192"/></a> 
+
-
<div>
+
-
<div id="Enlarge">
+
-
<a href="#" title="Enlarge"><img src="images/loading.gif" width="15" height="11" alt="" /></a></div>
+
-
<b>Figure 1.</b> Word "RNA" (from "<a href="#" rel="nofollow">http://www.mfpl.ac.at</a>")</div></div></div>
+
-
<p>By rationally mutate the Shine-Dalgarno (SD) and anti-Shine-Dalgarno (ASD) sequence, we are able to take advantage of the interaction of mRNA and ribosome to build our O-Key System of orthogonal ribosome and orthogonal mRNA. Within this system, we constructed an operon containing RFP and GFP coding sequence to verify the orthogonality of the O-Key. By selectively mutate the SD sequence of RFP or GFP, we were able to establish four translation pathways to characterize the effect of the O-Key System. In addition, we set up a model to predict the output of GFP and RFP under various circumstances. The model calculates the ΔG of ASD and SD sequence binding, and make use of this energy to evaluate the feasibility and translation efficiency of our O-Key System. The model turned out to be highly convincing as it corresponds with our wet lab result. In the end, both the wet and dry lab results matches our design. 
+
-
</p>
+
-
<br/><br/><br/><br/>
+
-
<h1> <span><span style="line-height:100%">Genetic Pollution Prevention and Genetic Encryption</span></span></h1>
+
-
<hr />
+
-
<div><div style="width:300px;"><a href="#"><img alt="" src="images/pic002.jpg" width="300" height="192" /></a>  <div><div id="Enlarge"><a href="#" title="Enlarge"><img src="images/loading.gif" width="15" height="11" alt="" /></a></div><b>Figure 2.</b> Comic of genetic pollution defence (from TJU iGEM Team 2012)</div></div></div>
+
-
<p>Aiming at preventing genetic pollution, we employed the O-Key System to establish a translational fence that can restrain unwanted protein expression. The convenience and effectiveness of the O-Key System will make it applied to a larger scale in genetic engineering. We predict different companies will embed the O-Key system in their various product to ensure biosafety. In the meantime, because the O-Key System includes a key and a lock, we can make use of this mechanism to encrypt information into cell or locking the product information. This characteristic showed a promising application in information encryption, intellectual property protection, etc. Furthermore, the O-Key System can be applied to the entire organism to construct an orthogonal organism. We began with the simplest creature - the phage, and worked on the RBS of its various protein. After mutation, the phage becomes a brand new orthogonal organism that can only infect the cells with orthogonal ribosomes.  Using this O-Key Phage, we greatly reduce risk of phage pollution in the lab, while performing regular experiment using the phage. At last, a successful interdisciplinary model that combines marketing and bioengineering was constructed to predict the diffusion of exogenous gene across space and time. This creative model used the analogy of human society and bacteria colony to predict the speed and probability of genetic transfer.</p><br/>
+
-
<h1> <span>Logic Metabolism Regulation</span></h1><hr />
+
-
<div ><div style="width:300px;"><a href="#"><img alt="" src="images/pic003.jpg" width="300" height="192"/></a>  <div>
+
-
<div id="Enlarge"><a href="#" title="Enlarge"><img src="images/loading.gif" width="15" height="11" alt="" /></a></div>
+
-
<b>Figure 3.</b> Metabolism Network (from TJU iGEM Team 2012)</div>
+
</div>
</div>
 +
 +
 +
</div>
</div>
</div>
-
<p>In this section, we describe the principles of Yeast Assembler, a novel way of assemble multiple fragments into a long operon, and specifically used this method to construct the gene needed to produce Violacein. The pathway of expressing violacein consists of five genes, and they build up a long operon. The conventional assembly methods for violacein takes too much time and labor, up to several weeks and offer resulting in failure, but using Yeast Assembler we can complete the whole process in a week. We will introduce and elaborate on the assembler in details. Through such an experiment, we could also prove the feasibilities of the O-Key System in regulating metabolism. Furthermore, we talked about the application of AND gate based on O-Key System in adjusting metabolism.
+
 
 +
<style type="text/css">
 +
.table1{font-family:Arial, Helvetica, sans-serif;width:100%;border-collapse:collapse;}
 +
.table1 td, .table1 th{font-size:14px;border:1px solid #ccc;padding:5px 10px 5px 10px;width:150px;}
 +
.table1 th {font-size:14px;text-align:left;padding-top:5px;padding-bottom:4px;background-color:#A7C942;color:#ffffff;}
 +
.table1 tr.alt td {color:#000000;background-color: #fafafa;}
 +
</style>
 +
<div class="main">
 +
<a name="anchor01" id="anchor01"></a>
 +
 
 +
</html>
 +
 
 +
=Luria Bertani Medium=
 +
 
 +
<html>
 +
 
 +
<hr /><br />
 +
<table class="table1">
 +
<tr>
 +
<td>Tryptone</td>
 +
<td>10g/L</td>
 +
</tr>
 +
 
 +
<tr class="alt">
 +
<td>Yeast extract</td>
 +
<td>5g/L</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>NaCl</td>
 +
<td>10g/L</td>
 +
</tr>
 +
 
 +
<tr class="alt">
 +
<td>&nbsp;</td>
 +
<td>&nbsp;</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td colspan="2"><b>Solid Luria Bertani medium: Add 15g/L Agar into Luria Bertani medium</b></td>
 +
 
 +
</tr>
 +
 
 +
</table>
 +
 
 +
<a name="anchor02" id="anchor02"></a>
 +
<br/>
 +
</html>
 +
 
 +
=M9 Medium=
 +
 
 +
<html>
 +
 
 +
<hr /><br />
 +
<table class="table1">
 +
<tr>
 +
<td>Na<sub>2</sub>HPO<sub>4</sub>&middot;12H<sub>2</sub>O</td>
 +
<td>15.1 g/L</td>
 +
</tr>
 +
 
 +
<tr class="alt">
 +
<td>KH<sub>2</sub>PO<sub>4</sub></td>
 +
<td>3 g/L</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>NaCl</td>
 +
<td>0.5 g/L</td>
 +
</tr>
 +
 
 +
<tr class="alt">
 +
<td>NH<sub>4</sub>Cl</td>
 +
<td>2 g/L</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>MgSO<sub>4</sub>&middot;7H<sub>2</sub>O</td>
 +
<td>0.25 g/L</td>
 +
</tr>
 +
 
 +
<tr class="alt">
 +
<td>CaCl<sub>2</sub></td>
 +
<td>11 mg/L</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>FeCl<sub>3</sub></td>
 +
<td>27 mg/L</td>
 +
</tr>
 +
 
 +
<tr class="alt">
 +
<td>ZnCl<sub>2</sub>&middot;4H<sub>2</sub>O</td>
 +
<td>2 mg/L</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Na<sub>2</sub>MoO<sub>4</sub>&middot;2H<sub>2</sub>O</td>
 +
<td>2 mg/L</td>
 +
</tr>
 +
 
 +
<tr class="alt">
 +
<td>CuSO<sub>4</sub></td>
 +
<td>1.9 mg/L</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>H<sub>3</sub>BO<sub>3</sub></td>
 +
<td>0.5 mg/L</td>
 +
</tr>
 +
 
 +
<tr class="alt">
 +
<td>Thiamine</td>
 +
<td>1 mg/L</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Bis-tris</td>
 +
<td>200 mmol/L</td>
 +
</tr>
 +
 
 +
<tr class="alt">
 +
<td>&nbsp;</td>
 +
<td>&nbsp;</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Glucose</td>
 +
<td>18g/L</td>
 +
</tr>
 +
 
 +
<tr class="alt">
 +
<td>&nbsp;</td>
 +
<td>&nbsp;</td>
 +
</tr>
 +
 
 +
</table>
 +
 
 +
<a name="anchor03" id="anchor03"></a>
 +
<br/>
 +
</html>
 +
 
 +
=Extracting Alkanes=
 +
 
 +
<html>
 +
 
 +
<hr /><br />
 +
 
 +
<p>1. Obtain 500μl sample in 1.5 ml microcentrifuge tube from 100ml medium.</p>
 +
<p>2. Add 500μl pure ethyl acetate into sample.</p>
 +
<p>3. Extract by adding 0.5mL of EthylAcetate, shaking at max speed for 10 min.</p>
 +
<p>4. Separate the water layer and EthylAcetate layer by centrifuge at 5000 rpm for 5min at 4℃</p>
 +
<p>5. Remove 300-400μl of the top Ethyl Acetate layer, filtering by membrane, and transfer to a 1.5 ml microcentrifuge tube.</p>
 +
<p>6. Store extracting sample at -20℃ bridge</p>
 +
 
 +
<a name="anchor04" id="anchor04"></a>
 +
<br/>
 +
</html>
 +
 
 +
=Ligation=
 +
 
 +
<html>
 +
<hr /><br />
 +
 
 +
<p>1.  Check the concentration of DNA fragments and vector which are going to be ligated.</p>
 +
<p>2.  Calcμlate  the  amount  of  part A/partB  and vector  added, based  on  the fragment length.  Note that a ligation using a molar ratio of 1:3-1:5 vector to inserts.</p>
 +
<p>3.  Add DNA/buffer and ligase together in the EP tube.<br /></p>
 +
<table class="table1">
 +
<tr>
 +
<td> Reaction system </td>
 +
<td>10.0μL </td>
 +
</tr>
 +
 
 +
<tr class="alt">
 +
<td> Part A </td>
 +
<td> A.0μL </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> Vector </td>
 +
<td> V.0μl </td>
 +
</tr>
 +
 
 +
<tr class="alt">
 +
<td>10x T4 Ligase Buffer </td>
 +
<td>1.0μL </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> T4 Ligase </td>
 +
<td>0.2μL </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td colspan="2">Add ddH2O until the total volume is 10.0μL</td>
 +
</tr>
 +
 
 +
</table>
 +
 
 +
<p>4.  Mix the reaction by pipetting up and down gently and microfuge briefly.</p>
 +
<p>5.  Incubate at 22°C for 40 min.</p>
 +
 
 +
<a name="anchor05" id="anchor05"></a>
 +
<br/>
 +
</html>
 +
 
 +
=Restriction Enzyme Digestion=
 +
 
 +
<html>
 +
<hr /><br />
 +
 
 +
<p>To check if the two selected restriction enzymes can perform effective catalysis in the same solution</p>
 +
<p>1. Mix DNA solution with the suitable amount of the master mix.<br />
 +
a.
 +
<table class="table1">
 +
<tr>
 +
<td> Reaction system </td>
 +
<td>10.0μL </td>
 +
</tr>
 +
 
 +
<tr class="alt">
 +
<td> DNA solution </td>
 +
<td>6.0μL </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>10x FD Buffer </td>
 +
<td>1.0μl </td>
 +
</tr>
 +
 
 +
<tr class="alt">
 +
<td> Each restriction enzyme </td>
 +
<td>0.2μL </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> ddH2O </td>
 +
<td>2.8μL </td>
 +
</tr>
 +
 
 +
</table>
 +
 
 +
b.
 +
<table class="table1">
 +
<tr>
 +
<td> Reaction system </td>
 +
<td>30.0μL </td>
 +
</tr>
 +
 
 +
<tr class="alt">
 +
<td> DNA solution </td>
 +
<td>3.0μL </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>10x FD Buffer </td>
 +
<td>3.0μl </td>
 +
</tr>
 +
 
 +
<tr class="alt">
 +
<td> Each restriction enzyme </td>
 +
<td>1.0μL </td>
 +
</tr>
 +
 
 +
<tr>
 +
<td> ddH2O </td>
 +
<td>23.0μL </td>
 +
</tr>
 +
 
 +
</table>
 +
 
</p>
</p>
 +
 +
<p>2.  Pipette up and down in the EP tube.</p>
 +
<p>3.  Incubate: 37°C for 30-40 min</p>
 +
 +
<a name="anchor06" id="anchor06"></a>
 +
<br/>
 +
</html>
 +
 +
=Competent Cell (e.g. E.coli BL 21)=
 +
 +
<html>
 +
<hr /><br />
 +
 +
<p>1.Inoculate 5μl BL21(Glycerol Storage) into 3 ml LB medium for an overnight cultures at 37 ℃ with 220rpm shaking</p>
 +
<p>2.Inoculate 50μl BL21 (from step 1) into 3 ml LB medium, inoculate a culture of 3ml LB medium, incubate for 2 h at 37℃,with 220rpm shaking</p>
 +
 +
<p>3.Harvest the bacteria cells by centrifuge at 5000 rpm in 1.5 ml microcentrifuge tube for 5min at 4℃</p>
 +
<p>4.Add 1 ml pre-cool 0.1M CaCl2 solution , mix bacteria cells by pipetting solution</p>
 +
<p>5. Place the microcentrifuge tube on ice for 20min</p>
 +
<p>6. Repeat step3 and step 4</p>
 +
<p>7. Harvest the bacteria cells by centrifuge at 5000 rpm in 1.5 ml microcentrifuge tube for 5min at 4℃</p>
 +
<p>8. Add 100 μl pre-cool 0.1M CaCl2 solution , mix bacteria cells by pipetting solution</p>
 +
<p>9. Place the microcentrifuge tube on ice for 20min</p>
 +
<p>10. Store Competent Cell at -80℃ bridge</p>
 +
 +
 +
 +
 +
<a name="anchor07" id="anchor07"></a>
 +
<br/>
 +
</html>
 +
 +
=DNA Agarose Gels=
 +
 +
<html>
 +
<hr /><br />
 +
 +
<p>1. Prepare a 1% weight-to-volume agarose gel(take 100ml as example)</p>
 +
<p>2. Dilute stock of 50×TAE to 1×with ddH2O.</p>
 +
 +
<p>3. Measure 100 ml of 1×TAE buffer.</p>
 +
<p>4. Transfer 1×TAE buffer to Erlenmeyer flask.</p>
 +
<p>5. Weigh out enough agarose to make 1% gel. (1% of 100mL is 1.0 g)</p>
 +
<p>6. Transfer agarose to Erlenmeyer flask.</p>
 +
<p>7. Melt agarose in microwave, stirring every 15-20 seconds until completely melted.</p>
 +
<p>8. Allow gel to cool until Erlenmeyer flask can be handled comfortably. Then add 1:20 volume ratio (5μl) GelRed Nucleic acid dye to the gel and shake the Erlenmeyer flask to dye the gel well.</p>
 +
<p>9. Pour agarose into gel tray, assemble gel pouring apparatus by inserting gate into slots.</p>
 +
 +
 +
<a name="anchor08" id="anchor08"></a>
 +
<br/>
 +
</html>
 +
 +
=Agarose Gel Electrophoresis=
 +
 +
<html>
 +
<hr /><br />
 +
 +
<p>1. Allow agarose to cool, place the gel in the apparatus rig with the wells facing the negative end (black-colored).</p>
 +
<p>2. Fill the rig with 1x TAE buffer.</p>
 +
 +
<p>3. Load 5µL of DNA maker into lane.</p>
 +
<p>4. Mix 1µL of 10x loading buffer with 10µL DNA sample, load them into lane.</p>
 +
<p>5. Run at 150V for 30 min.</p>
 +
<p>6. Use Gel imaging system check gel.</p>
 +
<p>7. Take picture for gel.</p>
 +
 +
 +
<a name="anchor09" id="anchor09"></a>
 +
<br/>
 +
</html>
 +
 +
=PCR Purification=
 +
 +
<html>
 +
<hr /><br />
 +
 +
<p>TIAN™quick Midi Purification Kit</p>
 +
<p>1. Balance the absorption column</p>
 +
 +
<p>a. Put the absorption column CB2 into the collection tube. Add 500 µl of the buffer BL to the absorption column CB2 and centrifuge at 12,000 rpm for 1 min at room temperature. Discard liquid and place the column back into the same collection tube.</p>
 +
<p>2. Add the buffer PB</p>
 +
<p>b. Determine the appropriate volume of the PCR reaction mixture.</p>
 +
<p>c. Add 5 times the volume of the buffer PB to the mixture and then mix by shaking or overtaxing the tube in increments.</p>
 +
<p>3. Absorption</p>
 +
<p>d. Apply the mixture to the column. For volumes greater than 800 µl, load the column and centrifuge successively, 800 μl at a time.</p>
 +
<p>e. Place the column at -20℃ for 5 min then centrifuge at 12,000 rpm for 1 min at room temperature.</p>
 +
<p>f. Apply the mixture in the collection tube to the column and then repeat step "e". Discard liquid and place the column back into the same collection tube.</p>
 +
<p>4. Wash</p>
 +
<p>g. Wash the column by adding 600μl of PW Wash Buffer diluted with absolute ethanol. Centrifuge at 12,000 rpm for 1 min at room temp.</p>
 +
<p>Note: PW Wash Buffer Concentrate must be diluted with absolute ethanol before use. See label for directions. If refrigerated, PW Wash Buffer must be brought to room temperature before use.</p>
 +
<p>h. Repeat step "g" with another 600μl of PW Wash Buffer diluted with absolute ethanol.</p>
 +
<p>I. Discard liquid and centrifuge the empty column for 2 min at 12,000 rpm to dry the column. Do not skip this step, it is critical for the removal of ethanol from the column.</p>
 +
<p>j. Place a column into a clean microcentrifuge tube. Then place the tube into the drying baker for 10min at 50℃ to dry the column matrix.</p>
 +
<p>5. Elution</p>
 +
<p>k. Add 50-70μl(depending on desired concentration of final product) of EB Buffer directly onto the column matrix.</p>
 +
<p>l. Incubate at 50℃ for 2 min. Centrifuge for 2 min at 12,000 rpm to elute DNA. </p>
 +
<p>m. Apply the mixture in the tube to the column and then repeat step "l" to yield any residual DNA.</p>
 +
 +
 +
 +
<a name="anchor10" id="anchor10"></a>
 +
<br/>
 +
</html>
 +
 +
=Gel Extraction of DNA (Spin Column Extraction)=
 +
 +
<html>
 +
<hr /><br />
 +
 +
<p>TIANgel Midi Purification Kit</p>
 +
<p>1.  Excise gel slice containing DNA fragment of interest. </p>
 +
 +
<p>a. Gel electrophoresis fractionates DNA fragments.</p>
 +
<p>b. The gel is exposed to UV to find the DNA fragments (stained by Ethidium bromide). </p>
 +
<p>c. The goal DNA band is identified.</p>
 +
<p>d.  Physically  remove  the  slice  of  gel  contains  the  goal  DNA  with  clean surgical blade.</p>
 +
<p>2.  DNA Purification</p>
 +
<p>e. Determine the appropriate volume of the gel slice by weighing it in a Clean 1.5 ml microcentrifuge tube.
 +
</p>
 +
<p>f. Add 3 times volume of Buffer PN more than the gel slice (0.1g gel account for 100μl). </p>
 +
<p>g.  Incubate  the  mixture  at  50°C  for  10  min  or  until  the  gel  has completely melted. </p>
 +
<p>h. Mix by shaking or overtaxing the tube in increments of 2 minutes.</p>
 +
<p>I.  Place a TIANGEN® DNA column CA2 in a provided 2 ml collection tube. Apply 500μl Buffer BL to the TIANGEN® DNA column, and centrifuge at 12,000 rpm for 1 min at room temperature. Discard liquid and place the TIANGEN® DNA column back into the same collection tube.</p>
 +
<p>j. Apply 700 μl of the DNA/agarose solution to the TIANGEN® DNA column, incubate at -25°C for 5 min and centrifuge at 12,000 rpm for 1 min at room temperature. </p>
 +
<p>k.  Put the liquid back into the TIANGEN® DNA column and redo j. Discard liquid and place the TIANGEN® DNA column back into the same collection tube. For volumes greater than 700 µ l, load the column and centrifuge successively, 700 μl at a time. Each TIANGEN® DNA column has a total capacity of 25μg DNA. If the expected yield is larger, divide the sample into the appropriate number of columns.</p>
 +
<p>l. Add 600 μl Buffer PW diluted with absolute ethanol into the TIANGEN® DNA column and incubate at room temperature for 2 min. Centrifuge at 12,000 rpm for 1 min at room temperature to wash the column. Discard the flow-through.</p>
 +
<p>Note:  Buffer PW Concentrate must be diluted with absolute ethanol before use.  See label for directions.  If refrigerated, Buffer PW must be brought to room temperature before use.</p>
 +
<p>m. Redo step l and re-use the collection tube. </p>
 +
<p>n. Centrifuge the empty TIANGEN® DNA column at 12,000 rpm for 2 min to dry the column. Do not skip this step, it is critical for the removal of ethanol from the TIANGEN® DNA column. </p>
 +
<p>o.  Place  the TANGEN®  DNA  column opened into  a  clean  1.5  ml  microcentrifuge  tube and incubate at 50 °C for at least 15 minute until there is no smell of ethanol.</p>
 +
<p>Add 50μl Buffer EB directly into the column and incubate at 50 °C for 5 minute. Centrifuge for 2 min at 12,000 rpm to elute DNA.  This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.</p>
 +
 +
<a name="anchor11" id="anchor11"></a>
 +
<br/>
 +
</html>
 +
 +
= Error -Prone PCR =
 +
 +
<html>
 +
<hr /><br />
 +
 +
<p>1. Make up a master mix of everything into one microcentrifuge tube.</p>
 +
<table width="100%" class="table1">
 +
<tr>
 +
<td> reaction system </td>
 +
<td>100.0μL </td>
 +
</tr>
 +
 +
<tr class="alt">
 +
<td>10x error Buffer(Mg<sup>-</sup>Mn<sup>-</sup>)</td>
 +
<td>10.0μL </td>
 +
</tr>
 +
 +
<tr>
 +
<td>100mM MgCl<sub>2</sub></td>
 +
<td>7μL </td>
 +
</tr>
 +
 +
<tr class="alt">
 +
<td>10mM MnCl<sub>2</sub></td>
 +
<td>5μL </td>
 +
</tr>
 +
 +
<tr>
 +
<td> dNTP </td>
 +
<td>8μL </td>
 +
</tr>
 +
 +
<tr class="alt">
 +
<td> dCTP </td>
 +
<td>8μL </td>
 +
</tr>
 +
 +
<tr>
 +
<td> dTTP </td>
 +
<td>8μL </td>
 +
</tr>
 +
 +
<tr class="alt">
 +
<td> Template </td>
 +
<td>1.0μL </td>
 +
</tr>
 +
 +
<tr>
 +
<td> Forward primer </td>
 +
<td>3.0μL </td>
 +
</tr>
 +
 +
<tr class="alt">
 +
<td> Reverse primer </td>
 +
<td>3.0μL </td>
 +
</tr>
 +
 +
<tr>
 +
<td> enzyme </td>
 +
<td>1μL </td>
 +
</tr>
 +
 +
<tr class="alt">
 +
<td> ddH2O </td>
 +
<td>46μL </td>
 +
</tr>
 +
 +
</table>
 +
 +
 +
<p>2. Pipette up and down in the microcentrifuge tube, drain or50.0μL solution to each PCR tube.</p>
 +
 +
<p>3. Run the "Error-Prone PCR" program, and adjust your extension time as described below.</p>
 +
<p> The "Error-Prone PCR" program</p>
 +
<p> Initial denaturation: 95°C for 5min </p>
 +
<p>25cycles of:</p>
 +
<p>95°C for 30 min </p>
 +
<p>55°C for 30min (different primers different annealing temperature)</p>
 +
<p>72°C for tmin (“t”depends on the length of goal sequence, 1minper 1000bp)</p>
 +
<p> Final extension: 72°C for 10 min</p>
 +
 +
<a name="anchor12" id="anchor12"></a>
 +
<br/>
 +
</html>
 +
 +
= ColonyPCR=
 +
 +
<html>
 +
<hr /><br />
 +
 +
<p>1. Make up a master mix of everything into one microcentrifuge tube.</p>
 +
 +
<table width="100%" class="table1">
 +
<tr>
 +
<td> reaction system </td>
 +
<td>10.0μL </td>
 +
</tr>
 +
 +
<tr class="alt">
 +
<td> dNTP </td>
 +
<td>1.0μL </td>
 +
</tr>
 +
 +
<tr>
 +
<td> Taq Buffer </td>
 +
<td>1.0μL </td>
 +
</tr>
 +
 +
<tr class="alt">
 +
<td> up primer </td>
 +
<td>0.2μL </td>
 +
</tr>
 +
 +
<tr>
 +
<td> down primer </td>
 +
<td>0.2μL </td>
 +
</tr>
 +
 +
<tr class="alt">
 +
<td> Enzyme(fast taq)</td>
 +
<td>0.1μL </td>
 +
</tr>
 +
 +
<tr>
 +
<td> ddH2O </td>
 +
<td>7.5μL </td>
 +
</tr>
 +
 +
</table>
 +
 +
<p>2.Run the "Simple PCR" program, and adjust your extension time as described below.</p>
 +
 +
<p> The "Simple PCR" program </p>
 +
<p> Initial denaturation: 95°C for 5min </p>
 +
<p>30cycles of:</p>
 +
<p>95°C for 30 min </p>
 +
<p>55°C for 30min (different primers different annealing temperature)</p>
 +
<p>72°C for tmin (“t”depends on the length of goal sequence, 1minper 1000bp)</p>
 +
<p> Final extension: 72°C for 10min</p>
 +
 +
<a name="anchor13" id="anchor13"></a>
 +
<br/>
 +
</html>
 +
 +
= Fluor Spectrophotometry=
 +
 +
<html>
 +
<hr /><br />
 +
 +
<p> 1.Obtain 1000μl sample in 1.5 ml microcentrifuge tube from a 3ml overnight cultures E.coli.</p>
 +
<p> 2.Harvest the bacteria cells by centrifuge at 8000 rpm in 1.5 ml microcentrifuge tube for 2min.</p>
 +
 +
<p>3. Add 1ml water, mix bacteria cells by pipetting solution </p>
 +
<p>4. Remove 200μL sample and add 2000μL water, measure the optical density(OD)</p>
 +
<p>5. Remove μL sample and add μL water, measure the fluorescence intensity.</p>
 +
<p>6. Calculate the fluorescence intensity of per OD sample, build the model</p>
 +
 +
 +
</div>
</div>
-
</div>
 
-
    </div>
 
-
<!--代码结束-->
 
</div>
</div>
 +
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<div style="display: block" id="goTopBtn">
<div style="display: block" id="goTopBtn">
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<a href="#top"><img border=0 src="images/lanren_top.jpg"></a>
+
<a href="#top"><img border=0 src="https://static.igem.org/mediawiki/2013/6/61/Top.jpg"></a>
</div>
</div>
</html>
</html>

Latest revision as of 21:20, 27 September 2013

Contents

Luria Bertani Medium



Tryptone 10g/L
Yeast extract 5g/L
NaCl 10g/L
   
Solid Luria Bertani medium: Add 15g/L Agar into Luria Bertani medium

M9 Medium



Na2HPO4·12H2O 15.1 g/L
KH2PO4 3 g/L
NaCl 0.5 g/L
NH4Cl 2 g/L
MgSO4·7H2O 0.25 g/L
CaCl2 11 mg/L
FeCl3 27 mg/L
ZnCl2·4H2O 2 mg/L
Na2MoO4·2H2O 2 mg/L
CuSO4 1.9 mg/L
H3BO3 0.5 mg/L
Thiamine 1 mg/L
Bis-tris 200 mmol/L
   
Glucose 18g/L
   

Extracting Alkanes



1. Obtain 500μl sample in 1.5 ml microcentrifuge tube from 100ml medium.

2. Add 500μl pure ethyl acetate into sample.

3. Extract by adding 0.5mL of EthylAcetate, shaking at max speed for 10 min.

4. Separate the water layer and EthylAcetate layer by centrifuge at 5000 rpm for 5min at 4℃

5. Remove 300-400μl of the top Ethyl Acetate layer, filtering by membrane, and transfer to a 1.5 ml microcentrifuge tube.

6. Store extracting sample at -20℃ bridge


Ligation



1. Check the concentration of DNA fragments and vector which are going to be ligated.

2. Calcμlate the amount of part A/partB and vector added, based on the fragment length. Note that a ligation using a molar ratio of 1:3-1:5 vector to inserts.

3. Add DNA/buffer and ligase together in the EP tube.

Reaction system 10.0μL
Part A A.0μL
Vector V.0μl
10x T4 Ligase Buffer 1.0μL
T4 Ligase 0.2μL
Add ddH2O until the total volume is 10.0μL

4. Mix the reaction by pipetting up and down gently and microfuge briefly.

5. Incubate at 22°C for 40 min.


Restriction Enzyme Digestion



To check if the two selected restriction enzymes can perform effective catalysis in the same solution

1. Mix DNA solution with the suitable amount of the master mix.
a.

Reaction system 10.0μL
DNA solution 6.0μL
10x FD Buffer 1.0μl
Each restriction enzyme 0.2μL
ddH2O 2.8μL
b.
Reaction system 30.0μL
DNA solution 3.0μL
10x FD Buffer 3.0μl
Each restriction enzyme 1.0μL
ddH2O 23.0μL

2. Pipette up and down in the EP tube.

3. Incubate: 37°C for 30-40 min


Competent Cell (e.g. E.coli BL 21)



1.Inoculate 5μl BL21(Glycerol Storage) into 3 ml LB medium for an overnight cultures at 37 ℃ with 220rpm shaking

2.Inoculate 50μl BL21 (from step 1) into 3 ml LB medium, inoculate a culture of 3ml LB medium, incubate for 2 h at 37℃,with 220rpm shaking

3.Harvest the bacteria cells by centrifuge at 5000 rpm in 1.5 ml microcentrifuge tube for 5min at 4℃

4.Add 1 ml pre-cool 0.1M CaCl2 solution , mix bacteria cells by pipetting solution

5. Place the microcentrifuge tube on ice for 20min

6. Repeat step3 and step 4

7. Harvest the bacteria cells by centrifuge at 5000 rpm in 1.5 ml microcentrifuge tube for 5min at 4℃

8. Add 100 μl pre-cool 0.1M CaCl2 solution , mix bacteria cells by pipetting solution

9. Place the microcentrifuge tube on ice for 20min

10. Store Competent Cell at -80℃ bridge


DNA Agarose Gels



1. Prepare a 1% weight-to-volume agarose gel(take 100ml as example)

2. Dilute stock of 50×TAE to 1×with ddH2O.

3. Measure 100 ml of 1×TAE buffer.

4. Transfer 1×TAE buffer to Erlenmeyer flask.

5. Weigh out enough agarose to make 1% gel. (1% of 100mL is 1.0 g)

6. Transfer agarose to Erlenmeyer flask.

7. Melt agarose in microwave, stirring every 15-20 seconds until completely melted.

8. Allow gel to cool until Erlenmeyer flask can be handled comfortably. Then add 1:20 volume ratio (5μl) GelRed Nucleic acid dye to the gel and shake the Erlenmeyer flask to dye the gel well.

9. Pour agarose into gel tray, assemble gel pouring apparatus by inserting gate into slots.


Agarose Gel Electrophoresis



1. Allow agarose to cool, place the gel in the apparatus rig with the wells facing the negative end (black-colored).

2. Fill the rig with 1x TAE buffer.

3. Load 5µL of DNA maker into lane.

4. Mix 1µL of 10x loading buffer with 10µL DNA sample, load them into lane.

5. Run at 150V for 30 min.

6. Use Gel imaging system check gel.

7. Take picture for gel.


PCR Purification



TIAN™quick Midi Purification Kit

1. Balance the absorption column

a. Put the absorption column CB2 into the collection tube. Add 500 µl of the buffer BL to the absorption column CB2 and centrifuge at 12,000 rpm for 1 min at room temperature. Discard liquid and place the column back into the same collection tube.

2. Add the buffer PB

b. Determine the appropriate volume of the PCR reaction mixture.

c. Add 5 times the volume of the buffer PB to the mixture and then mix by shaking or overtaxing the tube in increments.

3. Absorption

d. Apply the mixture to the column. For volumes greater than 800 µl, load the column and centrifuge successively, 800 μl at a time.

e. Place the column at -20℃ for 5 min then centrifuge at 12,000 rpm for 1 min at room temperature.

f. Apply the mixture in the collection tube to the column and then repeat step "e". Discard liquid and place the column back into the same collection tube.

4. Wash

g. Wash the column by adding 600μl of PW Wash Buffer diluted with absolute ethanol. Centrifuge at 12,000 rpm for 1 min at room temp.

Note: PW Wash Buffer Concentrate must be diluted with absolute ethanol before use. See label for directions. If refrigerated, PW Wash Buffer must be brought to room temperature before use.

h. Repeat step "g" with another 600μl of PW Wash Buffer diluted with absolute ethanol.

I. Discard liquid and centrifuge the empty column for 2 min at 12,000 rpm to dry the column. Do not skip this step, it is critical for the removal of ethanol from the column.

j. Place a column into a clean microcentrifuge tube. Then place the tube into the drying baker for 10min at 50℃ to dry the column matrix.

5. Elution

k. Add 50-70μl(depending on desired concentration of final product) of EB Buffer directly onto the column matrix.

l. Incubate at 50℃ for 2 min. Centrifuge for 2 min at 12,000 rpm to elute DNA.

m. Apply the mixture in the tube to the column and then repeat step "l" to yield any residual DNA.


Gel Extraction of DNA (Spin Column Extraction)



TIANgel Midi Purification Kit

1. Excise gel slice containing DNA fragment of interest.

a. Gel electrophoresis fractionates DNA fragments.

b. The gel is exposed to UV to find the DNA fragments (stained by Ethidium bromide).

c. The goal DNA band is identified.

d. Physically remove the slice of gel contains the goal DNA with clean surgical blade.

2. DNA Purification

e. Determine the appropriate volume of the gel slice by weighing it in a Clean 1.5 ml microcentrifuge tube.

f. Add 3 times volume of Buffer PN more than the gel slice (0.1g gel account for 100μl).

g. Incubate the mixture at 50°C for 10 min or until the gel has completely melted.

h. Mix by shaking or overtaxing the tube in increments of 2 minutes.

I. Place a TIANGEN® DNA column CA2 in a provided 2 ml collection tube. Apply 500μl Buffer BL to the TIANGEN® DNA column, and centrifuge at 12,000 rpm for 1 min at room temperature. Discard liquid and place the TIANGEN® DNA column back into the same collection tube.

j. Apply 700 μl of the DNA/agarose solution to the TIANGEN® DNA column, incubate at -25°C for 5 min and centrifuge at 12,000 rpm for 1 min at room temperature.

k. Put the liquid back into the TIANGEN® DNA column and redo j. Discard liquid and place the TIANGEN® DNA column back into the same collection tube. For volumes greater than 700 µ l, load the column and centrifuge successively, 700 μl at a time. Each TIANGEN® DNA column has a total capacity of 25μg DNA. If the expected yield is larger, divide the sample into the appropriate number of columns.

l. Add 600 μl Buffer PW diluted with absolute ethanol into the TIANGEN® DNA column and incubate at room temperature for 2 min. Centrifuge at 12,000 rpm for 1 min at room temperature to wash the column. Discard the flow-through.

Note: Buffer PW Concentrate must be diluted with absolute ethanol before use. See label for directions. If refrigerated, Buffer PW must be brought to room temperature before use.

m. Redo step l and re-use the collection tube.

n. Centrifuge the empty TIANGEN® DNA column at 12,000 rpm for 2 min to dry the column. Do not skip this step, it is critical for the removal of ethanol from the TIANGEN® DNA column.

o. Place the TANGEN® DNA column opened into a clean 1.5 ml microcentrifuge tube and incubate at 50 °C for at least 15 minute until there is no smell of ethanol.

Add 50μl Buffer EB directly into the column and incubate at 50 °C for 5 minute. Centrifuge for 2 min at 12,000 rpm to elute DNA. This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.


Error -Prone PCR



1. Make up a master mix of everything into one microcentrifuge tube.

reaction system 100.0μL
10x error Buffer(Mg-Mn-) 10.0μL
100mM MgCl2 7μL
10mM MnCl2 5μL
dNTP 8μL
dCTP 8μL
dTTP 8μL
Template 1.0μL
Forward primer 3.0μL
Reverse primer 3.0μL
enzyme 1μL
ddH2O 46μL

2. Pipette up and down in the microcentrifuge tube, drain or50.0μL solution to each PCR tube.

3. Run the "Error-Prone PCR" program, and adjust your extension time as described below.

The "Error-Prone PCR" program

Initial denaturation: 95°C for 5min

25cycles of:

95°C for 30 min

55°C for 30min (different primers different annealing temperature)

72°C for tmin (“t”depends on the length of goal sequence, 1minper 1000bp)

Final extension: 72°C for 10 min


ColonyPCR



1. Make up a master mix of everything into one microcentrifuge tube.

reaction system 10.0μL
dNTP 1.0μL
Taq Buffer 1.0μL
up primer 0.2μL
down primer 0.2μL
Enzyme(fast taq) 0.1μL
ddH2O 7.5μL

2.Run the "Simple PCR" program, and adjust your extension time as described below.

The "Simple PCR" program

Initial denaturation: 95°C for 5min

30cycles of:

95°C for 30 min

55°C for 30min (different primers different annealing temperature)

72°C for tmin (“t”depends on the length of goal sequence, 1minper 1000bp)

Final extension: 72°C for 10min


Fluor Spectrophotometry



1.Obtain 1000μl sample in 1.5 ml microcentrifuge tube from a 3ml overnight cultures E.coli.

2.Harvest the bacteria cells by centrifuge at 8000 rpm in 1.5 ml microcentrifuge tube for 2min.

3. Add 1ml water, mix bacteria cells by pipetting solution

4. Remove 200μL sample and add 2000μL water, measure the optical density(OD)

5. Remove μL sample and add μL water, measure the fluorescence intensity.

6. Calculate the fluorescence intensity of per OD sample, build the model

Retrieved from "http://2013.igem.org/Team:Tianjin/Protocol"