S-STEM Scholar Leilani Boren's Blog

 Introduction:     Due to the importance of preparing a protocol for future genetic sequences of members of the Deinococcus genus, further testing was done to evaluate methods of DNA extraction for high molecular weight. The primary focus this week was to utilize the kit that gave us a successful sequence of D. aquaticus  and modify that method for the future paper that does not require as high molecular weight in samples as does when evaluating isolates. D. aquaticus and D. radiodurans are effectively being used as standards in preparation for many different types within the genus. D. sonorensis  was also briefly evaluated this week due to it's strange plaque like structures that it forms. It is still hopeful that a chemical process, can be used without mechanical processing, in the Zymo kit. Above all else, it is imperative to accurately determine if results can be reproduced effectively using whatever protocol is ultimately decided upon.  Methods:  ...

 Introduction: Previous tests to alter the QUIAGEN protocol for high molecular weight DNA extraction to accommodate for the tough exterior of the Deinococcus   genus and specifically Deinococcus aquaticus  have been unsuccessful. It was decided due to a lack of confidence in Nanodrop readings to utilize the fluorometer going forward for testing samples post lysis in the protocol. It is hoped that the fluorometer will give more concrete insight as to how well the mechanical alternations in the protocol are effectively lysing the cells.  Methods:    Deinococcus aquaticus  was inoculated in ten milliliters of TGY broth media and allowed 48 hours for growth in an agitation incubator at thirty degrees Celsius. An OD600 reading was taken and the culture diluted with TGY broth in a separate tube for a total of one ml volume, and a secondary OD readings taken to ensure it was within protocol standards (0.05 to 0.3). These tubes were aliquoted into four a ...

 Introduction:     Previous experiments have been conducted using qPCR to evaluate the gene expression in Deinococcus radiodurans  for six target genes including acylase. lactonase, Met H, Met K, pfs, Lux S, and two reference genes, secA and GAP3. After two previous runs resulting in gene expressions in the treatment groups statistically identical to the control groups, it was determined that the 50 mM hydrogen peroxide solution may not be enough to induce the level of oxidative stress desired to witness changes in the gene expressions. A new run with the same target and reference genes will be conducted, using 100 mM hydrogen peroxide solution at the same 30 minute duration as previous trials. The first step in this process is to conduct a successful RNA extraction from the three treatment groups and three control groups. I did this process from start to finish by myself for the first time in order for Chad Albert to continue the process with cDNA synthesis and subs...

 Introduction:      Last week tests were done to evaluate different methods to achieve high molecular weight segments of DNA to be utilized in genetic sequencing. The data showed more improvements could be made to narrow down the exact specifications of ideal DNA extraction in terms of nanograms per microliter, and the gel being unsuccessful last week made it impossible to determine differences in size of segments between methods. The vast range between 30 seconds of inversion and two seconds of bead beating and 60 seconds of inversion and five seconds of bead beating needs to be confirmed in further experiments. A more comprehensive experiment was run testing a range of 30, 45 and 60 seconds inverting and 3, 4, and 5 seconds beat beating. Two more samples were run testing an RNA lysis buffer not a part of the original QIAGEN protocol, and just the lysozyme but incubated for an extended Peoria day of time. The goal this week is to run a successful gel that shows whet...