Evaluating DNA Extraction Methods Across Deinoccocus Species

Introduction;

    In the previous summer, one of the main projects taken by the lab was to formulate a DNA extraction protocol that could be used for 16 different Deinococcus species in order to sequence them and continue research. Not only did we do these species, but over 20 Deinococcus aquaticus isolates were also done, but with a greater focus on maintaining high molecular weight for the genomic DNA extracted. In doing this, the species were extracted by using different bead beating times depending on what was needed for each species and then the QIAGEN DNeasy kit, and the isolates with a much more intensive protocol, the QIAGEN genomic tip with some minor modifications, predominantly cell lysis by sonication. It was later found to be wit the new chemistries developed for sequencing that samples could not be eluted in anything other than nuclease free water, or else the sequence would be inhibited by a surplus of contaminants. Even the trace amounts of chemicals in elution buffers, including the elution buffer from the DNeasy kit and TE buffer used for the isolates, would render the samples nearly useless. Despite an effort to transfer the samples from elution buffer to water, it was determined that new DNA extractions were necessary in order to have hope of sequencing the species. This week, we attempted to apply the recently developed protocol for Deinococcus sonorensis to the other 15 species, and pivoted to an adaptation of the protocol that was developed for the aquaticus isolates and saw positive results that we believe should provide sufficient sequencing data. 


Methods:

    The cultures were originally grown on TGY plate and then inoculated 10 mL of TGY broth, and allowed to grow for 48 hours at 30 degrees Celsius at 100 RPM. 

    One mL of 48 hour culture was extracted and an OD600 value recorded prior to resuspending them in QIAGEN B1 lysis buffer, and sonicating the cells for a total 30 seconds with 5 seconds on, 5 seconds off at 12%. This lysate was then centrifuged, and supernatant used for either the ZYMO DNA cleanup and concentration kit or the rest of the QIAGEN protocol that included the RNase A and Proteinase K incubation and subsequent genomic tip column. Both were eluted in 50 ul of nuclease free water. 


Results:

    As can be seen in the data table, the QIAGEN genomic tip samples compared to the very same species originally done with the D. sonorensis protocol were both cleaner and had a much higher yield. The gram stains pictured are of each of the cultures that were used for the QIAGEN genomic tip protocol. 












Species

Ng/ul

260/280

260/230

Sonication and ZYMO DNA Cleanup and Conc. Kit

 

 

 

D. daejeonensis

32.9

1.86

1.68

D. grandis

5.89

1.77

1.13

D. pimensis

25.9

1.84

1.36

Sonication and QIAGEN Genomic Tip

 

 

 

D. daejeonensis

85.2

1.76

1.91

D. grandis

57.6

1.58

1.85

D. pimensis

92.8

1.80

1.94












Discussion: 

    It is clear to see that the adapted protocol that somehow worked well for D. sonorensis is not compatible with other species, and this discovery thankfully led to the success of the QIAGEN genomic tip protocol. The new sequencing cleanliness parameters have become more stringent, and the 260/280 value needing to be about 1.8 made the first protocol unable to compete, namely because decreasing the 260/280 to the ideal value of 1.8, it also directly decreased the 260/230 value, making the sample completely unacceptable. The QIAGEN genomic tip protocol thankfully pulled through and worked for three of the species so far, although D. grandis is not within parameters yet likely due to the slightly lower DNA yield, with 40 ul of sample left it is presumed that this sample can be cleaned up with the ZYMO cleanup kit and it will be completely usable for sequencing. Although the 260/230 would ideally be above 2.00, it is felt that singularly running these samples will be sufficient to collect useful data. It is yet to be seen while other species are grown and DNA extracted if they will also cooperate with the QIAGEN genomic tip protocol, or further adjustments will need to be made. 






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