Team:Groningen/Template/MODULE/Notebook/secretion/week5

From 2014.igem.org

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25 – 29 August
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August 25 - August 29
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<b>goal:</b> obtaining a construct with the promotors and GFP for promotor analysis in <i>L. lactis</i>.
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Obtaining constructs with different promoters and the gene coding for GFP for promoter analysis in <i>L. lactis</i>
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<div class="item">An overnight culture of <i>E. coli</i> DH5α with the P2 with RBS construct, pLas with RBS construct, and the gene coding for GFP with the double terminator construct was made</div>
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<div class="item">The constructs P2 with RBS, pLas with RBS, the gene coding for GFP with the double terminator and ssUSP45DspB were checked on gel, only P2 with RBS and pLas with RBS had the correct size</div>
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<div class="item">The same experiment with the ssUSP45DspB was performed again, but again no positive results were obtained</div>
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<div class="item">PCR was performed with the synthetic ssUSP45DspB gene, resulting in ssUSP45 and ssUSP45DspB without the His-tag</div>
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All the clones containing the constructs with p2+rbs, pLAS+rbs and gfp+Dterm were grown again to be mini-prepped. After this, these products, including ssUSP45DspB ran on a gel showing that p2+rbs and pLAS+rbs had the correct size, but ssUSP45DspB and gfp+Dterm did not have the correct size.
 
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Again, the Gblock of ssUSP45DspB was cloned with the digestion mix, and again rendering negative results.
 
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After a couple of failures with the small amount of gblocks which has been given, PCR was done on 1µl of the digestion mixture. The products which were produced were: ssUSP45 and ssUSP45DspB without His-tag.
 
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Revision as of 23:04, 17 October 2014

August 25 - August 29
 
Obtaining constructs with different promoters and the gene coding for GFP for promoter analysis in L. lactis
An overnight culture of E. coli DH5α with the P2 with RBS construct, pLas with RBS construct, and the gene coding for GFP with the double terminator construct was made
The constructs P2 with RBS, pLas with RBS, the gene coding for GFP with the double terminator and ssUSP45DspB were checked on gel, only P2 with RBS and pLas with RBS had the correct size
The same experiment with the ssUSP45DspB was performed again, but again no positive results were obtained
PCR was performed with the synthetic ssUSP45DspB gene, resulting in ssUSP45 and ssUSP45DspB without the His-tag
 
 
 
 
 

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