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<h3>RNA extraction</h3> | <h3>RNA extraction</h3> | ||
<p> | <p> | ||
− | We tested different methods to lyse cells and purify RNA. We compared these methods with commercial available kits. RNA purity was checked in denaturating urea gels or agarose gels. To quantify RNA after FINA extraction we transcribed it into cDNA | + | We tested different methods to lyse cells and purify RNA. We compared these methods with commercial available kits. RNA purity was checked in denaturating urea gels or agarose gels. To quantify RNA after FINA extraction we transcribed it into cDNA, amplified it using PCR, and then tried to quantify the products on an agarose gel. To test our system, we first used defined transcribed RNAs which mimicked our targets. After <i>in vitro</i> transcription, RNA was further purified using a phenol chloroform based method. </p> |
</td> | </td> | ||
</tr> | </tr> | ||
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<h3>Protein purification</h3> | <h3>Protein purification</h3> | ||
<p> | <p> | ||
− | We expressed our His-tagged proteins in <i>E.coli</i> strains and purified them using | + | We expressed our His-tagged proteins in <i>E.coli</i> strains and purified them using the Äkta purification system or Ni-NTA agarose. The latter had the advantage to incubate the cell lysate a certain time to get a better binding. To cleave off the His-SUMO or His-MBP tags from Cas13a proteins, we incubated them with the SUMO or TEV protease during dialysis over night, respectively. In some cases, we reloaded the cleaved protein solution again on Ni-NTA agarose to get rid of the thereby binding His-tag. The proteins were purified further via size exclusion chromatography. Protein purity was always checked in SDS PAGE gels.</p> |
<p> | <p> | ||
− | Cas13a Lbu is the central component of our diagnostic platform. The TEV Protease is part of our idea to the Intein-Extein readout, but apart from that, served as molecular tool for cleaving off the protein tags. A summary of all protein | + | Cas13a Lbu is the central component of our diagnostic platform. The TEV Protease is part of our idea to the Intein-Extein readout, but apart from that, served as molecular tool for cleaving off the protein tags. A summary of all protein information can also be found. |
</p> | </p> | ||
</td> | </td> | ||
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<h3>Readout</h3> | <h3>Readout</h3> | ||
<p> | <p> | ||
− | For our experiments, we chose Cas13a Lbu since it was reported to be the most active one. We first focused to get a running system and detect a fragment of <i>E.coli</i> 16S rRNA in plate reader experiments. We <i>in vitro</i> transcribed these target sequences, purified them using a phenol/chloroform based method and quantified resulting RNA in a denaturing SDS PAGE. As a reporter system we chose the RNase alert kit from IDT. Further testing included | + | For our experiments, we chose Cas13a Lbu since it was reported to be the most active one. We first focused to get a running system and detect a fragment of <i>E.coli</i> 16S rRNA in plate reader experiments. We <i>in vitro</i> transcribed these target sequences, purified them using a phenol/chloroform based method and quantified the resulting RNA in a denaturing SDS PAGE. As a reporter system we chose the RNase alert kit from IDT. Further testing included the detection of different targets, both bacteria and viruses. Next step was to detect RNA, which we isolated form actual bacterial sources. We then brought the system onto paper by lyophilization of our components. |
</p> | </p> | ||
</td> | </td> |
Revision as of 22:00, 31 October 2017