G.tigrina Hox gene DthoxC insertion into prokaryote E.coli / (by UNIamCloning)/2011/10/04

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PCR Check and Third DNA Isolation of G.tigrina

  • Today in lab we began by running a 1.0% agarose/TBE buffer electrophoresis gel to check success of last week's PCR amplification of our DthoxC planarian gene. The results were unsuccessful. The primers showed up at about 500 base pairs but between the 900-1000 bp range there was nothing which is around where we expected our gene to show. For another possible experiment, we were thinking of using sample Gt.3 again for another PCR but increasing the concentration of DNA (by increasing the amount of DNA used in each sample and decreasing the amount of water used in each sample).

We then measured out approximately 0.025g of G.tigrina - (whole organism will readily lyse in proteinase K and ATL buffer) - because this quantity is suggested by our DNA extraction kit, Qiagen DNeasy Blood & Tissue Kit - (for 20ul proteinase K and 180ul ATL buffer). This involved weighing out about 15-20 planarians, which is considerably more than we used for any previous extractions (1-2 organisms). The 0.025g sample was scraped into a 1.5mL tube with 20ul proteinase K and 180ul ATL buffer, vortexed, and placed into a 56°C water bath overnight. The sample will continue to be isolated and purified tomorrow, Wednesday 10/05/2011.