Team:ZJU-China/notebook.htm

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<h2>1 Stock solutions </h2>
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      <p>&nbsp;</p>
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<p>1.1 Ampicillin: 100 mg/mL. </p>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_1.htm">1 Stock solutions</a>
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<p>1 g Amp + 10mL water. Filter sterilize, freeze in aliquots.</p>
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<br>
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<p>&nbsp;</p>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_2.htm">2 Antibiotics</a>
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<p>1.2 Kanamycin: 50 mg/mL</p>
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<br>
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<p>0.5 g Kan + 10mL water. Filter sterilize, freeze in aliquots.</p>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_3.htm">3 Cell transformation</a>
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<p>&nbsp;</p>
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<p>1.3 Spectinomycin: 10 mg/mL</p>
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<p>0.1 g Amp + 10mL water. Filter sterilize, freeze in aliquots.</p>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_4.htm">4 Glycerol stock</a>
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<p>&nbsp;</p>
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<br>
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<p>1.4 Chloramphenicol:</p>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_5.htm">5 Miniprep</a>
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<p>&nbsp;</p>
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<br>
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<p>1.5 1M IPTG: </p>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_6.htm">6 DNA Agarose Gels</a>
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<p>2.4 grams +10mL Water. Filter sterilize, freeze in aliquots.</p>
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<p>&nbsp;</p>
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<p>1.6 50×TAE electrophoresis buffer:</p>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_7.htm">7 PCR</a>
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<p>Tris base 242 g</p>
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<br>
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<p>Glacial acetic acid 57.1ml</p>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_8.htm">8 PCR Purification</a>
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<p>Na2EDTA·2H2O 37.2 g</p>
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<br>
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<p>H2O to 1 liter</p>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_9.htm">9 Restriction Digest</a>
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<p>&nbsp;</p>
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<p>1.7 LB medium</p>
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<p>Tryptone 10 g</p>
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<p>Yeast Extract 5 g</p>
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<p>NaCl 10 g</p>
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<p>PH?</p>
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<p>Add H2O to 1L</p>
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<p>&nbsp;</p>
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<h2>2 Antibiotics</h2>
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<p>&nbsp;</p>
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<p>2.1 Ampicillin: 100 μg/mL</p>
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<p>&nbsp;</p>
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<p>2.2 Kanamycin: 50 μg/mL</p>
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<p>&nbsp;</p>
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<p>2.3 Spectinomycin: 100 μg/mL</p>
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<p>&nbsp;</p>
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<p>2.4 Chloramphenicol:</p>
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<p>&nbsp;</p>
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<p>2.5 For co- transformation: Ampicillin 50 μg/mL, Spectinomycin 25 μg/mL, Kanamycin 25 μg/mL</p>
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<p>&nbsp;</p>
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<h2>3 Cell transformation</h2>
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<p>&nbsp;</p>
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<p>3.1 Remove competent cells on ice to thaw.</p>
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<p>&nbsp;</p>
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<p>3.2 Add 1-10 μl of DNA to the cells. </p>
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<p>&nbsp;</p>
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<p>3.3 Incubate on ice for 20 min.</p>
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<p>&nbsp;</p>
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<p>3.4 Spread onto LB + antibiotic agar plate. Incubate overnight at 37℃. </p>
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<p>&nbsp;</p>
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<h2>4 Glycerol stock</h2>
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<p>&nbsp;</p>
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<p>4.1 Pick a colony from plate and grow them to OD600=0.6 in LB containing antibiotic.</p>
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<p>&nbsp;</p>
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<p>4.2 150 μl of glycerol add to 850 μl of cell suspension in freezing tube.</p>
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<p>&nbsp;</p>
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<p>4.3 Mix and freeze at -80℃.</p>
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<p>&nbsp;</p>
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<h2>5 Miniprep: AxyPrep Plasmid Miniprep Kit. Follow the manufacturer’s protocols.</h2>
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<p>&nbsp;</p>
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<h2>6 DNA Agarose Gels:</h2>
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<p>&nbsp;</p>
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<p>6.1 Weigh out 0.2g of agarose. Add 20mL of 1×TAE.</p>
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<p>&nbsp;</p>
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<p>6.2 Microwave until dissolved.</p>
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<p>&nbsp;</p>
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<p>6.3 cool to about 60℃. Add 2uL of 10,000×Gel Red.</p>
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<p>&nbsp;</p>
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<p>6.4 Pour gel slowly into the tank. Cool in cold room for 0.5 hour.</p>
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<p>&nbsp;</p>
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<h2>7 PCR:</h2>
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<p>&nbsp;</p>
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<p>7.1 Use TaKaRa Premix Taq Version 2.0. Follow the manufacturer’s protocols.</p>
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<p>&nbsp;</p>
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<p>7.2 Set up the following reaction in a PCR tube on ice.</p>
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<table>
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<tr>
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<td>Premix Taq</td>
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<td>12.5 &mu;l</td>
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</tr>
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<tr>
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<td>Template</td>
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<td>0.1 ng - 10 ng</td>
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</tr>
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<tr>
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<td>Primer F</td>
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<td>0.5 &mu;l</td>
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</tr>
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<tr>
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<td>Primer R</td>
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<td>0.5 &mu;l</td>
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</tr>
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<tr>
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<td>Nuclease-free water</td>
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<td>To 25 &mu;l</td>
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</tr>
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<tr>
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<td>Total volume</td>
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<td>25 &mu;l</td>
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</tr>
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</table>
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<p>7.3 PCR cycle:</p>
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<p>94℃      3min</p>
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<p>30cycles</p>
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<p>94℃      30s</p>
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<p>55℃      30s</p>
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<p>72℃      1min</p>
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<p>72℃      5min</p>
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<p>&nbsp;</p>
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<h2>8 PCR Purification: AxyPrep PCR Clearnup Kit. Follow the manufacturer’s protocols.</h2>
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<p>&nbsp;</p>
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<h2>9 Restriction Digest:</h2>
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<p>&nbsp;</p>
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<p>9.1 Instruction</p>
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<p>&nbsp;</p>
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<p>9.2 EroRI</p>
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<p>&nbsp;</p>
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<p>9.3 PstI</p>
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<p>&nbsp;</p>
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<p>9.4 ……</p>
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<p>&nbsp;</p>
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<h2>10 Gel Extraction</h2>
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<p>&nbsp;</p>
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<p>10.1 Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.</p>
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<p>&nbsp;</p>
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<p>10.2 Weigh the Gel.</p>
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<p>&nbsp;</p>
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<p>10.3 Use AxyPrep PCR Clearnup Kit. Follow the manufacturer’s protocols.</p>
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<p>&nbsp;</p>
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<h2>11 Ligation:</h2>
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<p>&nbsp;</p>
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<p>11.1 Use fermentas T4 DNA Ligase. Follow the manufacturer’s protocols.</p>
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<p>&nbsp;</p>
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<p>11.2 Set up the following reaction in a microcentrifuge tube on ice. </p>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_10.htm">10 Gel Extraction</a>
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<br>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_11.htm">11 Ligation</a>
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<br>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_12.htm">12 Growth Curve</a>
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<a target="proFrame" href="http://2012.igem.org/Team:ZJU-China/n_p_13.htm">13 Split GFP experiments</a>
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<table>
 
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<tr>
 
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<td>Linear vector DNA</td>
 
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<td>20 - 100ng</td>
 
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</tr>
 
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<tr>
 
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<td>Insert DNA</td>
 
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<td>10:1 molar ratio over vector</td>
 
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</tr>
 
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<tr>
 
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<td>10*T4 DNA Ligase Buffer</td>
 
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<td>2 &mu;l</td>
 
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</tr>
 
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<tr>
 
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<td>T4 DNA Ligase</td>
 
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<td>1 &mu;l</td>
 
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</tr>
 
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<tr>
 
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<td>Nuclease-free water</td>
 
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<td>To 20 &mu;l</td>
 
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</tr>
 
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<tr>
 
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<td>Total volume</td>
 
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<td>20 &mu;l</td>
 
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</tr>
 
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</table>
 
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<p>11.3 Incubate 30 min at 22℃.</p>
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<p>11.4 Heat inactivation of T4 DNA ligase at 65℃ for 10 min. (not necessary)</p>
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<p>&nbsp;</p>
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<iframe src="" frameborder="0" name="proFrame" width="100%" height="1000px"> </iframe>  
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<p>11.5 Use 10 μl of the mixture for transformation of 100μl competent cell.</p>
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<p>&nbsp;</p>
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<h2>12 Growth Curve:</h2>
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<p>&nbsp;</p>
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<p>12.1 Set up cells culture in ~2mL LB + antibiotic.</p>
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<p>&nbsp;</p>
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<p>12.2 Grow at 37℃, 250 rpm overnight.</p>
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<p>&nbsp;</p>
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<p>12.3 Dilute in LB + antibiotic so that final OD600 is ~0.05 for each culture.</p>
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<p>&nbsp;</p>
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<p>12.4 Take OD600 of freshly dilute cultures. Another dilution may be necessary.</p>
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<p>&nbsp;</p>
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<p>12.5 Grow at 37℃, 250 rpm.</p>
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<p>&nbsp;</p>
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<p>12.6 Take OD600 at 30mins.</p>
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<p>&nbsp;</p>
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<p>12.7 Once OD600 has reached ~0.1, begin taking ODs every 20mins.</p>
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<p>&nbsp;</p>
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<p>12.8 When OD600 =0.2, split cultures in half and induce half with 2mM IPTG.</p>
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<p>&nbsp;</p>
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<p>12.9 Once an OD 1.0 is reached dilute the culture 1:1 with media to take an accurate OD. Then multiply the OD value by the dilution factor.</p>
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<p>&nbsp;</p>
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<h2>13 Split GFP experiments</h2>
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<p>&nbsp;</p>
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<p>13.1 Co-transformation:</p>
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<p>13.1.1 Mix the plasmids of D0, FA and FB.</p>
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<p>13.1.2 Transform the mixed plasmids to BL21*(DE3)</p>
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<p>&nbsp;</p>
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<p>13.2 Pick a colony from the plate Co-transformation. Grow in LB(50 μg/mL Ampicillin, 25 μg/mL Spectinomycin, 25 μg/mL Kanamycin)</p>
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<p>&nbsp;</p>
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<p>13.3 Induce the cells by 0.2M IPTG for 2 hours at mid-log phase.</p>
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<p>&nbsp;</p>
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<p>13.4 Wash the cells twice with equivalent PBS</p>
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<p>&nbsp;</p>
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<p>13.5 Test with Synergy H1 Hybrid reader</p>
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<p>&nbsp;</p>
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<p>13.6 Take picture with Confocal Scanning Microscope.</p>
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Revision as of 21:07, 26 September 2012

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