Team:Slovenia/Notebook

From 2012.igem.org

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<h3>Restriction digest</h3>
<h3>Restriction digest</h3>
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<b>B. PCR product purification </b><br/>
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<p><b>PCR PRODUCT PURIFICATION </b></p>
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Desired PCR product was purified by GeneJet Gel Extraction Kit according to manufacturer's protocol.
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<p>Desired PCR products were purified by GeneJet Gel Extraction Kit according to the manufacturer's protocol. </p>
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<p><b>DNA CONCENTRATION. </b> An aliquot of the isolated DNA was analyzed using NanoDrop. </p>
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<h3>Gibson assembly </h3>
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<b>DNA concentration.</b> An aliquot of DNA isolation was analyzed using a NanoDrop.
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<p>Gibson assembly master mix was prepared according to the protocol published in Gibson et al., 2009. </p>
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<h3>Gibson Assembly</h3>
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Gibson assembly master mix was prepared as by Gibson at al., 2009.
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<li>50 ng of each PCR product were added to Gibson assembly master mix and incubated at 50 °C.</li>
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<li>50 ng of each PCR product was added to the Gibson assembly master mix and incubated at 50 °C 1h. </li>
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<li>After incubation, the transformation in bacteria was preformed.</li>
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<li>After incubation, the entire master mix volume was transformed into competent bacterial cells. </li>
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<h3>Culturing clones</h3>
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<h3> Culturing bacteria</h3>
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<b>A. BACTERIA</b><br/>
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<p>For plasmid DNA propagation two bacterial strains were used: <b>DH5alpha</b> [<i>fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17</i>] and <b>TOP10</b> [<i>mcrA, Δ(mrr-hsdRMS-mcrBC), Phi80lacZ(del)M15, ΔlacX74, deoR, recA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL(SmR), endA1,nupG</i>]. </p>
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For plasmid DNA propagation two bacterial strains were used: <b>DH5alpha</b> [fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17] and <b>TOP10</b> [mcrA, Δ(mrr-hsdRMS-mcrBC), Phi80lacZ(del)M15, ΔlacX74, deoR, recA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL(SmR), endA1,nupG].
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<p><b>Growth media for bacteria</b></p>
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<b>B. GROWTH MEDIA for BACTERIA</b><br/>
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<b>Luria Broth (LB)</b>
<b>Luria Broth (LB)</b>
<ul style="margin-left:15px;">  
<ul style="margin-left:15px;">  

Revision as of 16:35, 26 September 2012