Team:Hong Kong-CUHK/documentation.html

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</style>
</style>
<div id="safety-content" class="row">
<div id="safety-content" class="row">
     <!--Nav Bar -->
     <!--Nav Bar -->
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     <nav class="col-xs-3 bs-docs-sidebar">
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     <nav class="col-sm-3 hidden-xs bs-docs-sidebar">
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         <ul id="sidebar" class="nav nav-stacked fixed">
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         <ul id="sidebar" class="nav nav-stacked">
             <li class="active">
             <li class="active">
                 <a href="#protocol">Protocol</a>
                 <a href="#protocol">Protocol</a>
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                     <li><a href="#BL21">Preparation of chemically competent BL21 <br>E. coli cells</a>
                     <li><a href="#BL21">Preparation of chemically competent BL21 <br>E. coli cells</a>
                     </li>
                     </li>
-
<li><a href="primer">Primer Design</a>
+
                    <li><a href="primer">Primer Design</a>
                     </li>
                     </li>
-
<li><a href="#pcr1">PCR - Phusion DNA polymerase NEB</a>
+
                    <li><a href="#pcr1">PCR - Phusion DNA polymerase NEB</a>
                     </li>
                     </li>
-
<li><a href="#pcr2">PCR - Phusion DNA polymerase NEB</a>
+
                    <li><a href="#pcr2">PCR - PCR - LA Taq DNA polymerase Takara</a>
                     </li>
                     </li>
-
<li><a href="#BL21">Preparation of chemically competent BL21 E. coli cells</a>
+
                    <li><a href="#DDD">Double Digestion of DNA with 2 different restriction enzymes NEB</a>
 +
                    </li>
 +
                    <li><a href="#BL21">Preparation of chemically competent BL21 E. coli cells</a>
                     </li>
                     </li>
                 </ul>
                 </ul>
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             <li>
             <li>
                 <a href="#notebook">Notebook</a>
                 <a href="#notebook">Notebook</a>
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<ul class="nav nav-stacked">
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                <ul class="nav nav-stacked">
                     <li><a href="#june">June</a>
                     <li><a href="#june">June</a>
                     </li>
                     </li>
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                     <li><a href="#august">August</a>
                     <li><a href="#august">August</a>
                     </li>
                     </li>
-
<li><a href="#september">September</a>
+
                    <li><a href="#september">September</a>
                     </li>
                     </li>
                     <li><a href="#october">October</a>
                     <li><a href="#october">October</a>
                     </li>
                     </li>
                 </ul>
                 </ul>
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</li>
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            </li>
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         </ul>
         </ul>
     </nav>
     </nav>
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<!--Main Content -->
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    <!--Main Content -->
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<div class="col-xs-9">
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    <div class="col-sm-9">
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<section id="protocol" class="group">
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        <section id="protocol" class="group">
-
<h1>PROTOCOL<h1>
+
            <h2><b>PROTOCOL</b>
-
<h2 id="DNA"><b>Bacterial DNA extraction protocol for Azotobacter vinelandii or E.coli</b></h2>
+
            </h2>
-
<div class="subgroup well">We are using the <a href="http://www.takara.co.kr/file/manual/pdf/9763_e.v1309Da.pdf" target="_blank">TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit</a> of Takara according to the manufacturer directions.  
+
            <h3 id="DNA"><b>Bacterial DNA extraction protocol for Azotobacter vinelandii or E.coli</b>
-
</div>
+
            </h3>
-
<h2 id="DNA"><b>Miniprep</b></h2>
+
            <div class="subgroup well">We are using the <a href="http://www.takara.co.kr/file/manual/pdf/9763_e.v1309Da.pdf" target="_blank">TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit</a> of Takara according to the manufacturer directions.
-
<div class="subgroup well">We are using the <a href="http://eshop.intronbio.com/product/detail04.asp?pIdx=1" target="_target">DNA-spin&#8482 Plasmid DNA Purification Kit</a> of Intron Technology according to the manufacturer directions.  
+
            </div>
-
</div>
+
            <h3 id="miniprep"><b>Miniprep</b>
-
<h2 id="BL21"><b>Preparation of chemically competent BL21 E. coli cells</b></h2>
+
            </h3>
-
<div class="subgroup well">
+
            <div class="subgroup well">We are using the <a href="http://eshop.intronbio.com/product/detail04.asp?pIdx=1" target="_target">DNA-spin&#8482 Plasmid DNA Purification Kit</a> of Intron Technology according to the manufacturer directions.
-
<p>
+
            </div>
-
Day1<br>
+
            <h3 id="BL21"><b>Preparation of chemically competent BL21 E. coli cells</b>
-
Streak Bl21 on a LB agar plate without antibiotic, grow overnight in 37&#8451 incubator
+
            </h3>
-
</p>
+
            <div class="subgroup well">
 +
                <p>
 +
                    Day1
 +
                    <br>Streak Bl21 on a LB agar plate without antibiotic, grow overnight in 37&#8451 incubator
 +
                </p>
 +
 
 +
                <p>
 +
                    Day2
 +
                    <br>Pick a single colony and inoculate into 3ml LB broth, grow o/n in 37&#8451 shaker
 +
                    <br>Prepare & autoclave 500ml LB broth
 +
                    <br>Check if there is enough liquid nitrogen
 +
                    <br>
 +
                </p>
 +
                <p>Day3 Morning: pour the 3ml dense pre-culture into 500ml LB broth
 +
                    <br>Shake in 37C until OD600nm reach 0.8
 +
                    <br>[Melody's experience: it takes 4~5hrs]</p>
 +
 
 +
                <p>Solution Needed
 +
                    <br>Wash Buffer I
 +
                    <br>800mM MgCl2 + 20mM CaCl2</p>
-
<p>
+
                <p>Wash Buffer II
-
Day2<br>
+
                    <br>125mM CaCl2</p>
-
Pick a single colony and inoculate into 3ml LB broth, grow o/n in 37&#8451 shaker<br>
+
-
Prepare & autoclave 500ml LB broth<br>
+
-
Check if there is enough liquid nitrogen<br>
+
-
</p>
+
-
<p>Day3
+
-
Morning: pour the 3ml dense pre-culture into 500ml LB broth<br>
+
-
Shake in 37C until OD600nm reach 0.8<br>
+
-
[Melody's experience: it takes 4~5hrs]</p>
+
-
+
-
<p>Solution Needed<br>
+
-
Wash Buffer I<br>
+
-
800mM MgCl2 + 20mM CaCl2</p>
+
-
<p>Wash Buffer II<br>
+
                <p>Resuspend Buffer
-
125mM CaCl2</p>
+
                    <br>85mM CaCl2 + 15% glycerol [filtered]</p>
-
<p>Resuspend Buffer<br>
+
                <ol>
-
85mM CaCl2 + 15% glycerol [filtered]</p>
+
                    <li>pre-cool Wash Buffer I & Wash Buffer II in ice</li>
-
+
                    <li>Pre-cool the centrifuge to 4C (with fixed angle rotor)</li>
-
<ol>
+
                    <li>Check the OD600nm of the 500ml culture</li>
-
<li>pre-cool Wash Buffer I & Wash Buffer II in ice</li>
+
                    <li>Centrifuge the cells at 4000g for 5 mins, 4&#8451</li>
-
<li>Pre-cool the centrifuge to 4C (with fixed angle rotor)</li>
+
                    <li>Discard the supernatant</li>
-
<li>Check the OD600nm of the 500ml culture</li>
+
                    <li>Gently resuspend the pellet in 20ml ice cold Wash Buffer I</li>
-
<li>Centrifuge the cells at 4000g for 5 mins, 4&#8451</li>
+
                    <li>Put the samples on ice for 10 mins</li>
-
<li>Discard the supernatant </li>
+
                    <li>Centrifuge the cells at 4000g for 5 mins, 4&#8451</li>
-
<li>Gently resuspend the pellet in 20ml ice cold Wash Buffer I</li>
+
                    <li>Discard the supernatant</li>
-
<li>Put the samples on ice for 10 mins</li>
+
                    <li>Gently resuspend the pellet in 10ml ice cold Wash Buffer II</li>
-
<li>Centrifuge the cells at 4000g for 5 mins, 4&#8451</li>
+
                    <li>Put the samples on ice for 10 mins</li>
-
<li>Discard the supernatant </li>
+
                    <li>Centrifuge the cells at 4000g for 5 mins, 4&#8451</li>
-
<li>Gently resuspend the pellet in 10ml ice cold Wash Buffer II</li>
+
                    <li>Discard the supernatant</li>
-
<li>Put the samples on ice for 10 mins</li>
+
                    <li>Resuspend cells in 20ml ice cold Resuspend Buffer</li>
-
<li>Centrifuge the cells at 4000g for 5 mins, 4&#8451</li>
+
                    <li>Aliquot 200ul using sterile pre-chilled eppendorf tubes</li>
-
<li>Discard the supernatant </li>
+
                </ol>
-
<li>Resuspend cells in 20ml ice cold Resuspend Buffer</li>
+
            </div>
-
<li>Aliquot 200ul using sterile pre-chilled eppendorf tubes</li>
+
            <h3 id="primer"><b>Primer Design</b>
-
</ol>
+
            </h3>
-
</div>
+
            <div class="subgroup well">
-
<h2 id="primer"><b>Primer Design</b></h2>
+
                Primers were designed manually using <a href="http://www.bioinformatics.org/sms2/pcr_primer_stats.html" target="_blank">PCR Primer Stats</a> of Sequence Manipulation Suite, for analyzing secondary structures and also annealing conditions.
-
<div class="subgroup well">
+
            </div>
-
Primers were designed manually using <a href="http://www.bioinformatics.org/sms2/pcr_primer_stats.html" target="_blank">PCR Primer Stats</a> of Sequence Manipulation Suite, for analyzing secondary structures and also annealing conditions.
+
            <h3 id="pcr1"><b>PCR - Phusion DNA polymerase NEB</b>
-
</div>
+
            </h3>
-
<h2 id="pcr1"><b>PCR - Phusion DNA polymerase NEB</b></h2>
+
            <div class="subgroup well">
-
<div class="subgroup well">
+
                <p>50ul reaction</p>
-
<p>50ul reaction</p>
+
                <table class="table table-hover">
-
<table class="table table-hover">
+
                     <thead>
                     <thead>
                         <tr>
                         <tr>
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                         </tr>
                         </tr>
                     </thead>
                     </thead>
-
<tbody>
+
                    <tbody>
                         <tr>
                         <tr>
                             <td>DNA template</td>
                             <td>DNA template</td>
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                             <td>1.5</td>
                             <td>1.5</td>
                         </tr>
                         </tr>
-
<tr>
+
                        <tr>
                             <td>10uM Primer Fw</td>
                             <td>10uM Primer Fw</td>
                             <td>0.5</td>
                             <td>0.5</td>
                         </tr>
                         </tr>
-
<tr>
+
                        <tr>
                             <td>10uM Primer Rv</td>
                             <td>10uM Primer Rv</td>
                             <td>0.5</td>
                             <td>0.5</td>
                         </tr>
                         </tr>
-
<tr>
+
                        <tr>
                             <td>Phusion DNA polymerase</td>
                             <td>Phusion DNA polymerase</td>
                             <td>0.25-0.5</td>
                             <td>0.25-0.5</td>
-
                         </tr><tr>
+
                         </tr>
 +
                        <tr>
                             <td>100% DMSO</td>
                             <td>100% DMSO</td>
                             <td>1.5</td>
                             <td>1.5</td>
-
                         </tr><tr>
+
                         </tr>
 +
                        <tr>
                             <td>dH2O</td>
                             <td>dH2O</td>
                             <td>Up to 50</td>
                             <td>Up to 50</td>
                         </tr>
                         </tr>
-
<tr>
+
                        <tr>
-
<td><b>Total</b></td>
+
                            <td><b>Total</b>
-
<td><b>50</b></td>
+
                            </td>
-
</tr>
+
                            <td><b>50</b>
 +
                            </td>
 +
                        </tr>
                     </tbody>
                     </tbody>
                 </table>
                 </table>
-
 
-
<p>Cycling condition:<br>
 
-
Initial Denaturation (1 cycle): <br>
 
-
98°C 30 sec <br>
 
-
<br>
 
-
Amplification (35 cycles): <br>
 
-
98°C 10 sec <br>
 
-
55-72 °C 30 sec <br>
 
-
72°C 0.25- 0.5 min/kb <br>
 
-
<br>
 
-
Final elongation (1 cycle) <br>
 
-
72°C 3 min</p>
 
-
</div>
 
-
                <h2 id="pcr2"><b>PCR - LA Taq DNA polymerase Takara</b></h2>
+
                <p>Cycling condition:
-
<div class="subgroup well">
+
                    <br>Initial Denaturation (1 cycle):
-
<p>50ul reaction</p>
+
                    <br>98°C 30 sec
-
<table class="table table-hover">
+
                    <br>
 +
                    <br>Amplification (35 cycles):
 +
                    <br>98°C 10 sec
 +
                    <br>55-72 °C 30 sec
 +
                    <br>72°C 0.25- 0.5 min/kb
 +
                    <br>
 +
                    <br>Final elongation (1 cycle)
 +
                    <br>72°C 3 min</p>
 +
            </div>
 +
 
 +
            <h3 id="pcr2"><b>PCR - LA Taq DNA polymerase Takara</b>
 +
            </h3>
 +
            <div class="subgroup well">
 +
                <p>50ul reaction</p>
 +
                <table class="table table-hover">
                     <thead>
                     <thead>
                         <tr>
                         <tr>
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                         </tr>
                         </tr>
                     </thead>
                     </thead>
-
<tbody>
+
                    <tbody>
                         <tr>
                         <tr>
                             <td>DNA template</td>
                             <td>DNA template</td>
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                             <td>1.5</td>
                             <td>1.5</td>
                         </tr>
                         </tr>
-
<tr>
+
                        <tr>
                             <td>10uM Primer Fw</td>
                             <td>10uM Primer Fw</td>
                             <td>0.5</td>
                             <td>0.5</td>
                         </tr>
                         </tr>
-
<tr>
+
                        <tr>
                             <td>10uM Primer Rv</td>
                             <td>10uM Primer Rv</td>
                             <td>0.5</td>
                             <td>0.5</td>
                         </tr>
                         </tr>
-
<tr>
+
                        <tr>
                             <td>LA taq polymerase</td>
                             <td>LA taq polymerase</td>
                             <td>0.25-0.5</td>
                             <td>0.25-0.5</td>
-
                         </tr><tr>
+
                         </tr>
 +
                        <tr>
                             <td>100% DMSO</td>
                             <td>100% DMSO</td>
                             <td>1.5</td>
                             <td>1.5</td>
-
                         </tr><tr>
+
                         </tr>
 +
                        <tr>
                             <td>dH2O</td>
                             <td>dH2O</td>
                             <td>Up to 50</td>
                             <td>Up to 50</td>
                         </tr>
                         </tr>
-
<tr>
+
                        <tr>
-
<td><b>Total</b></td>
+
                            <td><b>Total</b>
-
<td><b>50</b></td>
+
                            </td>
-
</tr>
+
                            <td><b>50</b>
 +
                            </td>
 +
                        </tr>
 +
                    </tbody>
 +
                </table>
 +
 
 +
                <p>Cycling condition:
 +
                    <br>Initial Denaturation (1 cycle):
 +
                    <br>94°C 1 min
 +
                    <br>
 +
                    <br>Amplification (30 cycles):
 +
                    <br>98°C 10 sec
 +
                    <br>55-72 °C 30 sec
 +
                    <br>68°C 0.5- 1 min/kb
 +
                    <br>
 +
                    <br>Final elongation (1 cycle)
 +
                    <br>72°C 5 min</p>
 +
            </div>
 +
            <h3>NEB Gibson assembly</h3>
 +
            <div class="subgroup well">
 +
                <p>We are using the Gibson Assembly® Master Mix of NEB Inc. according to the manufacturer directions.</p>
 +
            </div>
 +
            <br>
 +
 
 +
            <h3 id="DDD"><b>Double Digestion of DNA with 2 different restriction enzymes NEB</b>
 +
            </h3>
 +
            <div class="subgroup well">
 +
                <p>30μl reaction</p>
 +
                <table class="table table-hover">
 +
                    <thead>
 +
                        <tr>
 +
                            <th>Reactives</th>
 +
                            <th>Volume(μL)</th>
 +
                        </tr>
 +
                    </thead>
 +
                    <tbody>
 +
                        <tr>
 +
                            <td>DNA</td>
 +
                            <td>Up to 1 μg</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>CutSmart™ Buffer</td>
 +
                            <td>3</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>EcoRI-HF®*</td>
 +
                            <td>1</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>SpeI/PstI/XbaI*</td>
 +
                            <td>1</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td>dH2O</td>
 +
                            <td>Up to 30μL</td>
 +
                        </tr>
 +
                        <tr>
 +
                            <td><b>Total</b>
 +
                            </td>
 +
                            <td><b>30</b>
 +
                            </td>
 +
                        </tr>
                     </tbody>
                     </tbody>
                 </table>
                 </table>
-
+
                <p>Incubate at 37 °C incubator or heat bath for 0.25 to 2 hours.
-
<p>Cycling condition:<br>
+
                    <br>*For combinations of restriction enzymes other than the above, please kindly refer to Double Digest Finder from NEB Inc. for suitable buffer condition.</p>
-
Initial Denaturation (1 cycle): <br>
+
                <br>
-
94°C 1 min <br>
+
                <h3 id="dna"><b>DNA ligation with T4 DNA ligase NEB</b>
-
<br>
+
                </h3>
-
Amplification (30 cycles): <br>
+
                <div class="subgroup well">
-
98°C 10 sec <br>
+
                    <table class="table table-hover">
-
55-72 °C 30 sec <br>
+
                        <thead>
-
68°C 0.5- 1 min/kb <br>
+
                            <tr>
-
<br>
+
                                <th>Reactives</th>
-
Final elongation (1 cycle) <br>
+
                                <th>Volume(μL)</th>
-
72°C 5 min</p>
+
                            </tr>
-
</div>
+
                        </thead>
 +
                        <tbody>
 +
                            <tr>
 +
                                <td>T4 DNA ligase</td>
 +
                                <td>1</td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>Buffer 10X</td>
 +
                                <td>2</td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>Vector DNA</td>
 +
                                <td>n pmol</td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>Insert DNA</td>
 +
                                <td>3n pmol</td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td>dH2O</td>
 +
                                <td>Up to 20μL</td>
 +
                            </tr>
 +
                            <tr>
 +
                                <td><b>Total</b>
 +
                                </td>
 +
                                <td><b>20</b>
 +
                                </td>
 +
                            </tr>
 +
                        </tbody>
 +
                    </table>
 +
                    <p>Incubate at room temperature for 0.25 -1 hours.</p>
 +
                </div>
 +
        </section>
-
</section>
+
        <section id="notebook" class="group">
 +
            <h2><b>NOTEBOOK</b>
 +
            </h2>
 +
            <h3 id="june"><b>JUNE</b>
 +
            </h3>
 +
            <div class="subgroup well">
 +
                <ol>
 +
                    <li>Preparation of Competent Cells of dh5&#945 and BL21, aliquot the competent cells, stored in liquid nitrogen and then -80C refrigerator.</li>
 +
                    <li>Transformation of: J23100-119 into dh5&#945</li>
 +
                    <li>Preparation of special medium and plate for A. vinelandii</li>
 +
                    <li>Make LB agar solution</li>
 +
                    <br>
-
<section id="notebook" class="group">
+
                    <li>Study the growth curve characteristics of A. vinelandii</li>
-
<h1><b>NOTEBOOK</b></h1>
+
                    <li>Study the natural antibiotics resistance of A. vinelandii</li>
-
<h2 id="june"><b>JUNE</b></h2>
+
                    <br>
-
<div class="subgroup well">
+
                    <li>Make plate with antibiotics -Ampicillin, kanamycin and chloramphenicol.</li>
-
<ol>
+
                    <li>Preparation of Competent Cells of A. vinelandii</li>
-
<li>Preparation of Competent Cells of dh5&#945 and BL21, aliquot the competent cells, stored in liquid nitrogen and then -80C refrigerator.</li>
+
                    <li>Purchase for primers for the Carbon fixation system project.</li>
-
<li>Transformation of: J23100-119 into dh5&#945</li>
+
                    <li>Primer dilution of the all carbon fixation system primers</li>
-
<li>Preparation of special medium and plate for A. vinelandii</li>
+
                    <li>Amplification by using PCR, following by run gel of A. vinelandii nifB gene.</li>
-
<li>Make LB agar solution</li>
+
                    <li>Amplification by using PCR, following by run gel of A. vinelandii nifQ gene.</li>
-
<br>
+
                    <br>
-
<li>Calibrate pH valve for N.P. and H.S. special medium</li>
+
                    <li>Amplification by using PCR, following by run gel of A. vinelandii FdxA gene.</li>
-
<li>Study the growth curve characteristics of A. vinelandii</li>
+
                    <li>Amplification by using PCR, following by run gel of Aquifex aeolicus mbhS3 gene.</li>
-
<li>Study the natural antibiotics resistance of A. vinelandii</li>
+
                    <li>Amplification by using PCR, following by run gel of Aquifex aeolicus mbhL3 gene.</li>
-
<br>
+
                    <li>Amplification by using PCR, following by run gel of E. coli SH3PDZ domain gene.</li>
-
<li>Make plate with antibiotics -Ampicillin, kanamycin and chloramphenicol.</li>
+
                </ol>
-
<li>Preparation of Competent Cells of A. vinelandii</li>
+
            </div>
-
<li>Purchase for primers for the Carbon fixation system project.</li>
+
-
<li>Primer dilution of the all carbon fixation system primers </li>
+
-
<li>Amplification by using PCR, following by run gel of A. vinelandii nifB gene.</li>
+
-
<li>Amplification by using PCR, following by run gel of A. vinelandii nifQ gene.</li>
+
-
<br>
+
-
<li>Amplification by using PCR, following by run gel of A. vinelandii FdxA gene. </li>
+
-
<li>Amplification by using PCR, following by run gel of Aquifex aeolicus mbhS3 gene.</li>
+
-
<li>Amplification by using PCR, following by run gel of Aquifex aeolicus mbhL3 gene.</li>
+
-
<li>Amplification by using PCR, following by run gel of E. coli SH3PDZ domain gene.</li>
+
-
</ol>
+
-
</div>
+
-
<h2><b>JULY</b></h2>
+
            <h3 id="july"><b>JULY</b>
-
<div class="subgroup well">
+
            </h3>
-
<ol>
+
            <div class="subgroup well">
-
<li>Make plate with 2% hard agar with antibiotics- chloramphenicol.</li>
+
                <ol>
-
<li>Make plate with antibiotics -Ampicillin.</li>
+
                    <li>Make plate with 2% hard agar with antibiotics- chloramphenicol.</li>
-
<li>Overlapping PCR of A. vinelandii nifB gene, A. vinelandii nifQ gene and A. vinelandii FdxA gene</li>
+
                    <li>Make plate with antibiotics -Ampicillin.</li>
-
<li>Restriction of Overlapping PCR product A. vinelandii nifB-nifQ-FdA, ligase into double terminator plasmid and transform into dh5&#945.</li>
+
                    <li>Overlapping PCR of A. vinelandii nifB gene, A. vinelandii nifQ gene and A. vinelandii FdxA gene</li>
-
<li>Overlapping PCR of Aquifex aeolicus mbhS3 gene and mbhL3 gene.</li>
+
                    <li>Restriction of Overlapping PCR product A. vinelandii nifB-nifQ-FdA, ligase into double terminator plasmid and transform into dh5&#945.</li>
-
<li>Restriction of Overlapping PCR product Aquifex aeolicus mbhS3-L3, ligase into double terminator plasmid and transform into dh5&#945.</li>
+
                    <li>Overlapping PCR of Aquifex aeolicus mbhS3 gene and mbhL3 gene.</li>
-
<li>Make LB agar solution</li>
+
                    <li>Restriction of Overlapping PCR product Aquifex aeolicus mbhS3-L3, ligase into double terminator plasmid and transform into dh5&#945.</li>
-
<li>Restriction of PCR product E. coli SH3PDZ domain gene, ligase into C backbone and transform into dh5&#945.</li>
+
                    <li>Make LB agar solution</li>
-
<li>Stable genome integration of J23100 into A. vinelandii and study its characteristics</li>
+
                    <li>Restriction of PCR product E. coli SH3PDZ domain gene, ligase into C backbone and transform into dh5&#945.</li>
-
<li>Purchase for primers for the Nitrogen- repressible T7 expression system.</li>
+
                    <li>Stable genome integration of J23100 into A. vinelandii and study its characteristics</li>
-
<li>Arrival of synthetic sequences of A. vinelandii nitrogenase structural gene</li>
+
                    <li>Purchase for primers for the Nitrogen- repressible T7 expression system.</li>
-
<li>Try Gibson assembly of synthetic sequences of A. vinelandii nitrogenase structural gene</li>
+
                    <li>Arrival of synthetic sequences of A. vinelandii nitrogenase structural gene</li>
-
</ol>
+
                    <li>Try Gibson assembly of synthetic sequences of A. vinelandii nitrogenase structural gene</li>
-
</div>
+
                </ol>
 +
            </div>
-
<h2><b>AUGUST</b></h2>
+
            <h3 id="august"><b>AUGUST</b>
-
<div class="subgroup well">
+
            </h3>
-
<ol>
+
            <div class="subgroup well">
-
<li>Arrival of synthetic sequences of A. vinelandii nitrogenase accessory gene.</li>
+
                <ol>
-
<li>Arrival of synthetic sequences of Aquifex aeolicus hydrogenase accessory gene.</li>
+
                    <li>Arrival of synthetic sequences of A. vinelandii nitrogenase accessory gene.</li>
-
<li>Arrival of all primers for the Nitrogen- repressible T7 expression system </li>
+
                    <li>Arrival of synthetic sequences of Aquifex aeolicus hydrogenase accessory gene.</li>
-
<li>Primer dilution of the all Nitrogen- repressible T7 expression system primers </li>
+
                    <li>Arrival of all primers for the Nitrogen- repressible T7 expression system</li>
-
<li>Amplification by using PCR, following by run gel of A. vinelandii nifH gene.</li>
+
                    <li>Primer dilution of the all Nitrogen- repressible T7 expression system primers</li>
-
<li>Amplification by using PCR, following by run gel of A. vinelandii nifK gene</li>
+
                    <li>Amplification by using PCR, following by run gel of A. vinelandii nifH gene.</li>
-
<li>Amplification by using PCR, following by run gel of A. vinelandii nifH gene with strong rbs.</li>
+
                    <li>Amplification by using PCR, following by run gel of A. vinelandii nifK gene</li>
-
<li>Amplification by using PCR, following by run gel of A. vinelandii nifH gene with weak rbs.</li>
+
                    <li>Amplification by using PCR, following by run gel of A. vinelandii nifH gene with strong rbs.</li>
-
<li>Transformation of T7 RNA polymerase and T7 promoter into dh5&#945.</li>
+
                    <li>Amplification by using PCR, following by run gel of A. vinelandii nifH gene with weak rbs.</li>
-
<li>Amplification by using PCR, following by run gel of BL21 T7 RNA polymerase.</li>
+
                    <li>Transformation of T7 RNA polymerase and T7 promoter into dh5&#945.</li>
-
<li>Overlapping PCR of A. vinelandii nifH gene with strong rbs and BL21 T7 RNA polymerase.</li>
+
                    <li>Amplification by using PCR, following by run gel of BL21 T7 RNA polymerase.</li>
-
<li>Overlapping PCR of A. vinelandii nifH gene with weak rbs and BL21 T7 RNA polymerase.</li>
+
                    <li>Overlapping PCR of A. vinelandii nifH gene with strong rbs and BL21 T7 RNA polymerase.</li>
-
<li>Amplification by using oligo PCR, following by run gel of random sequence</li>
+
                    <li>Overlapping PCR of A. vinelandii nifH gene with weak rbs and BL21 T7 RNA polymerase.</li>
-
<li>Transformation of ptet- mRFP-d.t. and mRFP-d.t. into dh5&#945.</li>
+
                    <li>Amplification by using oligo PCR, following by run gel of random sequence</li>
-
<li>Amplification by using oligo PCR, following by run gel of part of mRFP</li>
+
                    <li>Transformation of ptet- mRFP-d.t. and mRFP-d.t. into dh5&#945.</li>
-
<li>Restriction of PCR product random sequence, ligase into ptet- mRFP-d.t. plasmid and transform into dh5&#945.</li>
+
                    <li>Amplification by using oligo PCR, following by run gel of part of mRFP</li>
-
<li>Transformation of amilcp-d.t. into dh5&#945.</li>
+
                    <li>Restriction of PCR product random sequence, ligase into ptet- mRFP-d.t. plasmid and transform into dh5&#945.</li>
-
<li>Restriction of PCR product nifH, ligase into mRFP-d.t. plasmid and transform into dh5&#945.</li>
+
                    <li>Transformation of amilcp-d.t. into dh5&#945.</li>
-
</ol>
+
                    <li>Restriction of PCR product nifH, ligase into mRFP-d.t. plasmid and transform into dh5&#945.</li>
-
</div>
+
                </ol>
 +
            </div>
-
<h2><b>September</b></h2>
+
            <h3 id="september"><b>September</b>
-
<div class="subgroup well">
+
            </h3>
-
<ol>
+
            <div class="subgroup well">
-
<li>Try Gibson Assembly of synthetic sequences of A. vinelandii nitrogenase accessory gene.</li>
+
                <ol>
-
<li>Try Gibson Assembly of synthetic sequences of Aquifex aeolicus hydrogenase accessory gene.</li>
+
                    <li>Try Gibson Assembly of synthetic sequences of A. vinelandii nitrogenase accessory gene.</li>
-
<li>Restriction of PCR product A. vinelandii nifH gene with strong rbs and BL21 T7 RNA polymerase, ligase into double terminator plasmid and transform into dh5&#945.</li>
+
                    <li>Try Gibson Assembly of synthetic sequences of Aquifex aeolicus hydrogenase accessory gene.</li>
-
<li>Restriction of PCR product A. vinelandii nifH gene with weak rbs and BL21 T7 RNA polymerase, ligase into double terminator plasmid and transform into dh5&#945.</li>
+
                    <li>Restriction of PCR product A. vinelandii nifH gene with strong rbs and BL21 T7 RNA polymerase, ligase into double terminator plasmid and transform into dh5&#945.</li>
-
<li>Restriction of biobrick A. vinelandii nifH gene with strong rbs and BL21 T7 RNA polymerase-double terminator ligase into random sequence with ptet-mRFP-d.t. and transform into dh5&#945.</li>
+
                    <li>Restriction of PCR product A. vinelandii nifH gene with weak rbs and BL21 T7 RNA polymerase, ligase into double terminator plasmid and transform into dh5&#945.</li>
-
<li>Restriction of biobrick A. vinelandii nifH gene with weak rbs and BL21 T7 RNA polymerase-double terminator ligase into random sequence with ptet-mRFP-d.t. and transform into dh5&#945.</li>
+
                    <li>Restriction of biobrick A. vinelandii nifH gene with strong rbs and BL21 T7 RNA polymerase-double terminator ligase into random sequence with ptet-mRFP-d.t. and transform into dh5&#945.</li>
-
<li>Restriction of PCR product A. vinelandii nifK gene,ligase into biobrick A. vinelandii nifH gene with strong rbs and BL21 T7 RNA polymerase-double terminator and random sequence with ptet-mRFP-d.t. and transform into dh5&#945.</li>
+
                    <li>Restriction of biobrick A. vinelandii nifH gene with weak rbs and BL21 T7 RNA polymerase-double terminator ligase into random sequence with ptet-mRFP-d.t. and transform into dh5&#945.</li>
-
<li>Restriction of PCR product A. vinelandii nifK gene,ligase into biobrick A. vinelandii nifH gene with weak rbs and BL21 T7 RNA polymerase-double terminator and random sequence with ptet-mRFP-d.t. and transform into dh5&#945.</li>
+
                    <li>Restriction of PCR product A. vinelandii nifK gene,ligase into biobrick A. vinelandii nifH gene with strong rbs and BL21 T7 RNA polymerase-double terminator and random sequence with ptet-mRFP-d.t. and transform into dh5&#945.</li>
-
<li>Restriction of PCR product A. vinelandii nifK gene, ligase into C back bone and transform into dh5&#945.</li>
+
                    <li>Restriction of PCR product A. vinelandii nifK gene,ligase into biobrick A. vinelandii nifH gene with weak rbs and BL21 T7 RNA polymerase-double terminator and random sequence with ptet-mRFP-d.t. and transform into dh5&#945.</li>
-
<li>Restriction of PCR product A. vinelandii nifH gene, ligase into C back bone and transform into dh5&#945.</li>
+
                    <li>Restriction of PCR product A. vinelandii nifK gene, ligase into C back bone and transform into dh5&#945.</li>
-
<li>Restriction of PCR product part of mRFP, ligase into C back bone and transform into dh5&#945.</li>
+
                    <li>Restriction of PCR product A. vinelandii nifH gene, ligase into C back bone and transform into dh5&#945.</li>
-
<li>Restriction of BBa_K1314013, ligase into A backbone and transform into dh5&#945.</li>
+
                    <li>Restriction of PCR product part of mRFP, ligase into C back bone and transform into dh5&#945.</li>
-
<li>Restriction of BBa_K1314014, ligase into A backbone and transform into dh5&#945.</li>
+
                    <li>Restriction of BBa_K1314013, ligase into A backbone and transform into dh5&#945.</li>
-
<li>Restriction of PCR product part of mRFP, ligase into amilcp- d.t. and transform into dh5&#945.</li>
+
                    <li>Restriction of BBa_K1314014, ligase into A backbone and transform into dh5&#945.</li>
-
<li>Restriction of PCR product, random sequence, ligase into T7 promoter and transform into dh5 &#945.</li>
+
                    <li>Restriction of PCR product part of mRFP, ligase into amilcp- d.t. and transform into dh5&#945.</li>
-
<li>Restriction of biobrick random sequence- T7 promoter, ligase into C backbone and transform into dh5&#945</li>
+
                    <li>Restriction of PCR product, random sequence, ligase into T7 promoter and transform into dh5 &#945.</li>
-
<li>Restriction of biobrick random sequence- T7 promoter, ligase into amilcp- d.t.-part of mRFP, and transform into dh5&#945.</li>
+
                    <li>Restriction of biobrick random sequence- T7 promoter, ligase into C backbone and transform into dh5&#945</li>
-
<li>Send all biobricks to sequencing</li>
+
                    <li>Restriction of biobrick random sequence- T7 promoter, ligase into amilcp- d.t.-part of mRFP, and transform into dh5&#945.</li>
-
<li>Transformation of BBa_K1314011 into BL21.</li>
+
                    <li>Send all biobricks to sequencing</li>
-
<li>Stable genome integration of BBa_K1314013 into A. vinelandii</li>
+
                    <li>Transformation of BBa_K1314011 into BL21.</li>
-
<li>Stable genome integration of BBa_K1314014 into A. vinelandii</li>
+
                    <li>Stable genome integration of BBa_K1314013 into A. vinelandii</li>
-
<li>Stable genome integration of BBa_K1314011 into A. vinelandii</li>
+
                    <li>Stable genome integration of BBa_K1314014 into A. vinelandii</li>
-
<li>Blunt end ligation and transformation to make BBa_K1314015</li>
+
                    <li>Stable genome integration of BBa_K1314011 into A. vinelandii</li>
-
</ol>
+
                    <li>Blunt end ligation and transformation to make BBa_K1314015</li>
-
</div>
+
                </ol>
 +
            </div>
-
<h2><b>October</b></h2>
+
            <h3 id="october"><b>October</b>
-
<div class="subgroup well">1. Submission of all biobricks to the registry</div>
+
            </h3>
-
</section>
+
            <div class="subgroup well">1. Submission of all biobricks to the registry</div>
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       <section id="protocol" class="group">

PROTOCOL

Bacterial DNA extraction protocol for Azotobacter vinelandii or E.coli

We are using the <a href="http://www.takara.co.kr/file/manual/pdf/9763_e.v1309Da.pdf" target="_blank">TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit</a> of Takara according to the manufacturer directions.

Miniprep

We are using the <a href="http://eshop.intronbio.com/product/detail04.asp?pIdx=1" target="_target">DNA-spin&#8482 Plasmid DNA Purification Kit</a> of Intron Technology according to the manufacturer directions.

Preparation of chemically competent BL21 E. coli cells

Day1
Streak Bl21 on a LB agar plate without antibiotic, grow overnight in 37&#8451 incubator

Day2
Pick a single colony and inoculate into 3ml LB broth, grow o/n in 37&#8451 shaker
Prepare & autoclave 500ml LB broth
Check if there is enough liquid nitrogen

Day3 Morning: pour the 3ml dense pre-culture into 500ml LB broth
Shake in 37C until OD600nm reach 0.8
[Melody's experience: it takes 4~5hrs]

Solution Needed
Wash Buffer I
800mM MgCl2 + 20mM CaCl2

Wash Buffer II
125mM CaCl2

Resuspend Buffer
85mM CaCl2 + 15% glycerol [filtered]

  1. pre-cool Wash Buffer I & Wash Buffer II in ice
  2. Pre-cool the centrifuge to 4C (with fixed angle rotor)
  3. Check the OD600nm of the 500ml culture
  4. Centrifuge the cells at 4000g for 5 mins, 4&#8451
  5. Discard the supernatant
  6. Gently resuspend the pellet in 20ml ice cold Wash Buffer I
  7. Put the samples on ice for 10 mins
  8. Centrifuge the cells at 4000g for 5 mins, 4&#8451
  9. Discard the supernatant
  10. Gently resuspend the pellet in 10ml ice cold Wash Buffer II
  11. Put the samples on ice for 10 mins
  12. Centrifuge the cells at 4000g for 5 mins, 4&#8451
  13. Discard the supernatant
  14. Resuspend cells in 20ml ice cold Resuspend Buffer
  15. Aliquot 200ul using sterile pre-chilled eppendorf tubes

Primer Design 

               Primers were designed manually using <a href="http://www.bioinformatics.org/sms2/pcr_primer_stats.html" target="_blank">PCR Primer Stats</a> of Sequence Manipulation Suite, for analyzing secondary structures and also annealing conditions.

PCR - Phusion DNA polymerase NEB

50ul reaction

<thead> </thead> <tbody> </tbody>
Reactives Volume(μL)
DNA template 1
5x Phusion HF Buffer 10
10mM dNTPs 1.5
10uM Primer Fw 0.5
10uM Primer Rv 0.5
Phusion DNA polymerase 0.25-0.5
100% DMSO 1.5
dH2O Up to 50
Total 50

Cycling condition:
Initial Denaturation (1 cycle):
98°C 30 sec

Amplification (35 cycles):
98°C 10 sec
55-72 °C 30 sec
72°C 0.25- 0.5 min/kb

Final elongation (1 cycle)
72°C 3 min

PCR - LA Taq DNA polymerase Takara

50ul reaction

<thead> </thead> <tbody> </tbody>
Reactives Volume(μL)
DNA template 1
10x LA taq Buffer 5
10mM dNTPs 1.5
10uM Primer Fw 0.5
10uM Primer Rv 0.5
LA taq polymerase 0.25-0.5
100% DMSO 1.5
dH2O Up to 50
Total 50

Cycling condition:
Initial Denaturation (1 cycle):
94°C 1 min

Amplification (30 cycles):
98°C 10 sec
55-72 °C 30 sec
68°C 0.5- 1 min/kb

Final elongation (1 cycle)
72°C 5 min

NEB Gibson assembly

We are using the Gibson Assembly® Master Mix of NEB Inc. according to the manufacturer directions.

Double Digestion of DNA with 2 different restriction enzymes NEB

30μl reaction

<thead> </thead> <tbody> </tbody>
Reactives Volume(μL)
DNA Up to 1 μg
CutSmart™ Buffer 3
EcoRI-HF®* 1
SpeI/PstI/XbaI* 1
dH2O Up to 30μL
Total 30

Incubate at 37 °C incubator or heat bath for 0.25 to 2 hours.
*For combinations of restriction enzymes other than the above, please kindly refer to Double Digest Finder from NEB Inc. for suitable buffer condition.

DNA ligation with T4 DNA ligase NEB

<thead> </thead> <tbody> </tbody>
Reactives Volume(μL)
T4 DNA ligase 1
Buffer 10X 2
Vector DNA n pmol
Insert DNA 3n pmol
dH2O Up to 20μL
Total 20

Incubate at room temperature for 0.25 -1 hours.

       </section>

       <section id="notebook" class="group">

NOTEBOOK

JUNE

  1. Preparation of Competent Cells of dh5&#945 and BL21, aliquot the competent cells, stored in liquid nitrogen and then -80C refrigerator.
  2. Transformation of: J23100-119 into dh5&#945
  3. Preparation of special medium and plate for A. vinelandii
  4. Make LB agar solution
    5.
  5. Study the growth curve characteristics of A. vinelandii
  6. Study the natural antibiotics resistance of A. vinelandii
    7.
  7. Make plate with antibiotics -Ampicillin, kanamycin and chloramphenicol.
  8. Preparation of Competent Cells of A. vinelandii
  9. Purchase for primers for the Carbon fixation system project.
  10. Primer dilution of the all carbon fixation system primers
  11. Amplification by using PCR, following by run gel of A. vinelandii nifB gene.
  12. Amplification by using PCR, following by run gel of A. vinelandii nifQ gene.
    13.
  13. Amplification by using PCR, following by run gel of A. vinelandii FdxA gene.
  14. Amplification by using PCR, following by run gel of Aquifex aeolicus mbhS3 gene.
  15. Amplification by using PCR, following by run gel of Aquifex aeolicus mbhL3 gene.
  16. Amplification by using PCR, following by run gel of E. coli SH3PDZ domain gene.

JULY

  1. Make plate with 2% hard agar with antibiotics- chloramphenicol.
  2. Make plate with antibiotics -Ampicillin.
  3. Overlapping PCR of A. vinelandii nifB gene, A. vinelandii nifQ gene and A. vinelandii FdxA gene
  4. Restriction of Overlapping PCR product A. vinelandii nifB-nifQ-FdA, ligase into double terminator plasmid and transform into dh5&#945.
  5. Overlapping PCR of Aquifex aeolicus mbhS3 gene and mbhL3 gene.
  6. Restriction of Overlapping PCR product Aquifex aeolicus mbhS3-L3, ligase into double terminator plasmid and transform into dh5&#945.
  7. Make LB agar solution
  8. Restriction of PCR product E. coli SH3PDZ domain gene, ligase into C backbone and transform into dh5&#945.
  9. Stable genome integration of J23100 into A. vinelandii and study its characteristics
  10. Purchase for primers for the Nitrogen- repressible T7 expression system.
  11. Arrival of synthetic sequences of A. vinelandii nitrogenase structural gene
  12. Try Gibson assembly of synthetic sequences of A. vinelandii nitrogenase structural gene


AUGUST

  1. Arrival of synthetic sequences of A. vinelandii nitrogenase accessory gene.
  2. Arrival of synthetic sequences of Aquifex aeolicus hydrogenase accessory gene.
  3. Arrival of all primers for the Nitrogen- repressible T7 expression system
  4. Primer dilution of the all Nitrogen- repressible T7 expression system primers
  5. Amplification by using PCR, following by run gel of A. vinelandii nifH gene.
  6. Amplification by using PCR, following by run gel of A. vinelandii nifK gene
  7. Amplification by using PCR, following by run gel of A. vinelandii nifH gene with strong rbs.
  8. Amplification by using PCR, following by run gel of A. vinelandii nifH gene with weak rbs.
  9. Transformation of T7 RNA polymerase and T7 promoter into dh5&#945.
  10. Amplification by using PCR, following by run gel of BL21 T7 RNA polymerase.
  11. Overlapping PCR of A. vinelandii nifH gene with strong rbs and BL21 T7 RNA polymerase.
  12. Overlapping PCR of A. vinelandii nifH gene with weak rbs and BL21 T7 RNA polymerase.
  13. Amplification by using oligo PCR, following by run gel of random sequence
  14. Transformation of ptet- mRFP-d.t. and mRFP-d.t. into dh5&#945.
  15. Amplification by using oligo PCR, following by run gel of part of mRFP
  16. Restriction of PCR product random sequence, ligase into ptet- mRFP-d.t. plasmid and transform into dh5&#945.
  17. Transformation of amilcp-d.t. into dh5&#945.
  18. Restriction of PCR product nifH, ligase into mRFP-d.t. plasmid and transform into dh5&#945.


September

  1. Try Gibson Assembly of synthetic sequences of A. vinelandii nitrogenase accessory gene.
  2. Try Gibson Assembly of synthetic sequences of Aquifex aeolicus hydrogenase accessory gene.
  3. Restriction of PCR product A. vinelandii nifH gene with strong rbs and BL21 T7 RNA polymerase, ligase into double terminator plasmid and transform into dh5&#945.
  4. Restriction of PCR product A. vinelandii nifH gene with weak rbs and BL21 T7 RNA polymerase, ligase into double terminator plasmid and transform into dh5&#945.
  5. Restriction of biobrick A. vinelandii nifH gene with strong rbs and BL21 T7 RNA polymerase-double terminator ligase into random sequence with ptet-mRFP-d.t. and transform into dh5&#945.
  6. Restriction of biobrick A. vinelandii nifH gene with weak rbs and BL21 T7 RNA polymerase-double terminator ligase into random sequence with ptet-mRFP-d.t. and transform into dh5&#945.
  7. Restriction of PCR product A. vinelandii nifK gene,ligase into biobrick A. vinelandii nifH gene with strong rbs and BL21 T7 RNA polymerase-double terminator and random sequence with ptet-mRFP-d.t. and transform into dh5&#945.
  8. Restriction of PCR product A. vinelandii nifK gene,ligase into biobrick A. vinelandii nifH gene with weak rbs and BL21 T7 RNA polymerase-double terminator and random sequence with ptet-mRFP-d.t. and transform into dh5&#945.
  9. Restriction of PCR product A. vinelandii nifK gene, ligase into C back bone and transform into dh5&#945.
  10. Restriction of PCR product A. vinelandii nifH gene, ligase into C back bone and transform into dh5&#945.
  11. Restriction of PCR product part of mRFP, ligase into C back bone and transform into dh5&#945.
  12. Restriction of BBa_K1314013, ligase into A backbone and transform into dh5&#945.
  13. Restriction of BBa_K1314014, ligase into A backbone and transform into dh5&#945.
  14. Restriction of PCR product part of mRFP, ligase into amilcp- d.t. and transform into dh5&#945.
  15. Restriction of PCR product, random sequence, ligase into T7 promoter and transform into dh5 &#945.
  16. Restriction of biobrick random sequence- T7 promoter, ligase into C backbone and transform into dh5&#945
  17. Restriction of biobrick random sequence- T7 promoter, ligase into amilcp- d.t.-part of mRFP, and transform into dh5&#945.
  18. Send all biobricks to sequencing
  19. Transformation of BBa_K1314011 into BL21.
  20. Stable genome integration of BBa_K1314013 into A. vinelandii
  21. Stable genome integration of BBa_K1314014 into A. vinelandii
  22. Stable genome integration of BBa_K1314011 into A. vinelandii
  23. Blunt end ligation and transformation to make BBa_K1314015


October

1. Submission of all biobricks to the registry
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