Team:Heidelberg/Delftibactin/DelRest

From 2013.igem.org

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                                   <h1>Week 11</h1>
                                   <h1>Week 11</h1>
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                                   <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">
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During the past week we managed to amplify 11 kb as a single fragment including the genes DelA to DelF from genomic DNA from <i>D. Acidovorans</i>. Furthermore we were able to equip our biobrick backbone pSB4K5 with the desired overlaps to the genes of the Delftibactin cluster. We also succeeded in the amplification of the very last gene in our construct, DelL. However still a lot of work has to be done, since the amplification of DelF-G and DelO-P turned out to be more complicated. Since our initial plan, to amplify DelO-P with a Gibson primer which includes an overlaps to DelL and furthermore introduces an artificial ribosom binding site, turned out to be quite ambitious, we orderd shorter versions of all our Gibson primers, used to amplify genomic DNA. Using these shorter primers which should anneal better we will try to preamplify the desired genes in order to obtain a specific template for the amplification with the needed overlaps. Furthermore we will optimize the PCR conditions for the amplification of DelF-G.
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During the past week we managed to amplify a single fragment of 11 kbp including the genes DelA to DelF from genomic DNA of <i>D. Acidovorans</i>. Furthermore we were able to equip our biobrick backbone pSB4K5 with the desired overlaps to the genes of the Delftibactin cluster. We also succeeded in the amplification of the very last gene in our construct, DelL. However still a lot of work has to be done, since the amplification of DelF-G and DelO-P turned out to be more complicated. Since our initial plan, to amplify DelO-P with a Gibson primer which includes an overlaps to DelL and furthermore introduces an artificial ribosom binding site, turned out to be quite ambitious, we orderd shorter versions of all our Gibson primers, used to amplify genomic DNA. Using these shorter primers which should anneal better we will try to preamplify the desired genes in order to obtain a specific template for the amplification with the needed overlaps. Furthermore we will optimize the PCR conditions for the amplification of DelF-G.
                                   </p>
                                   </p>

Revision as of 19:33, 2 October 2013

Del Rest. Creating a 32 kbp plasmid.

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