Difference between revisions of "Team:Uppsala/Transcriptomics/PolyA Tailing"

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<p>Throughout the worm buster project we have at many times felt the need for assistance or input on different parts of our project. Without those small but immensely meaningful things our project would never had gotten this far and in the worse case never been possible at all. Therefore we would like to express our gratitude to all the people that supported us throughout the project. </p>  
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<p>Poly(A)-tails are a series of adenosine (A) nucleotides that are assembled in a string at the 3’ end of the mRNA, like a tail. Poly(A)-tails are important in the cells for both stabilising and signaling, marking the mRNA as ready to be used for different purposes. In eukaryotic cells, Poly(A)-tails are made naturally in the cell for most mRNA strands. In prokaryotes however, the tails are shorter, more uncommon, and in many cases such as in E.coli, there are no tails on most mRNA at all.<br><br>
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Because poly(A)-tails are a long sequence of the same nucleotide, they make good targets for primers of genes with otherwise unknown sequences. In the next step, we will be using primers that will bind to poly(A)-tails and thus, we need to synthesize them onto our RNA samples.</p>
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<h2>Experiment</h2>
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<p>The purified RNA sample retrieved from previous mRNA purification step is solved in nuclease free water and ready to use. The key reagent is the poly(A) polymerase, an enzyme that attach the adenosine nucleotides onto the mRNA that is directly added into the sample. <br><br>
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As you might already know no work is done for free, and that is the case for the poly(A) polymerase. Thereby ATP is added, which is an energy molecule that activates the poly(A) polymerase. The mRNA is then analyzed with gel-electrophoresis to confirm that the poly(A) tail attachment was a success.</p>
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<h2>Result</h2>
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<p>A successful poly(A) tail have a complete chain of adenosines connected to the 3’UTR of the mRNA strand. The attachment is analyzed with gel-electrophoresis and a successful attachment looks like our results in <b>figure 1</b>.
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                            <p><b>Figure 1:</b> A successful poly(A) tail attachment onto the mRNA strand, where the ladder shows specific lengths of the mRNA according to the table. The longer mRNA strand you have the longer poly(A) tail you have succeeded to attach.</p>
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                          <img src=”https://static.igem.org/mediawiki/2018/0/07/T--Uppsala--Transcriptomics-PolyA-Gel.jpg”>   
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Revision as of 08:56, 15 October 2018