Restarting AMC and Creating set qPCR Procedures

     After the conference we took a few days, spending some time cleaning up from the project and discussing some if the things we weren't able to get to like discussing the gram stains as well as looking into the UV comparison more as some of the other groups didn't get to finish their comparison. For the gram stains we theorized that the cells started to die earlier causing gram stains done two or so days after to give false results. We also theorized that the R2A UV results may have been different if we didn't plate on R2A again after UV exposure, as it may have starved the struggling cells. 

    With the characterization project effectively over for us, we were tasked with coming up with a set qPCR procedure, including set reference genes. Because we need to test each of the reference genes in deinococcus we need to perform qPCR on all of them, however from the original list of recommended reference genes we were able to narrow it down. Looking at "Table 1 Selected candidate reference genes, their corresponding product name, primer sequences (annealing temperature of 60 °C), amplicon size in base pairs (pb), their respective PCR amplification efficiencies and the mean CT values (± standard deviation) assessed in A. baumannii cells submitted to various experimental conditions and at different phases of growth." from de Oliveira, P.A.A., Baboghlian, J., Ramos, C.O.A. et al. Selection and validation of reference genes suitable for gene expression analysis by Reverse Transcription Quantitative real-time PCR in Acinetobacter baumannii. Sci Rep. 

    We were able to narrow down the full list down to era, fabD, ftsZ, groEL, rho, ropB and rpoD. This was done by selecting only those with a standard deviation of +/- .7 or lower. However era and ftsZ do not have the standard deviation data so we may end up cutting those. We also looked into each of the genes and where they come from/what they are associated with in order to get a better idea of each gene. 

    For the qPCR procedure itself we have decided to continue with the Luna Universal One-Step RT-qPCR Kit Protocol as it seems to be the most efficient for the RNA to cDNA conversion.

    In terms of the AMC project, both the side MetK and main project, we are picking both back up. There were some issues with the E. coli and D. caeni samples that have delayed the RNA isolations as the samples we have are both struggling to grow and we will need to get fresh samples from freeze back. Instead we have spent more time trying to gain a deeper understanding of the biochemical/organic chemistry that is happening, as well as looking into the qPCR procedures and both the recommended reference genes and chosen AMC reference genes.   






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