Preparing DNA Samples from Multiple Deinococcus species for Sequencing

 Introduction:

    After weeks of trying to adjust the protocol to optimize the DNA extraction process, it was decided this week that the best option would be to attempt to transfer the original samples from last summer that are currently in TE buffer, as discussed previously is unsuitable for sequencing since it contains trace amounts of EDTA, to water. The fear in doing this is that some samples will fall out, predominantly by not having enough DNA input to the ZYMO DNA cleanup and concentrator kit which has been used in the past. It seems as though if some specie samples do fall out the best course of action will be to go back to bead beating lysis methods with the DNeasy kit by QIAGEN, but we would like to avoid this as much as possible to prevent even further issues with sample extraction and time. Unfortunately time is a critical factor because the sequencing equipment is only useful for a certain number of days before it is ineffective for sequencing, so the fewer species that has to be redone the better for this project. This week five different species were attempted to transfer them from TE to PCR water, using the ZYMO DNA cleanup kit, with some mixed results.  

Methods:

    Each sample was measured using a pipette for approximate volume prior to beginning the ZYMO DNA cleanup and concentrator kit.

   A 2:1 ratio was used with DNA binding buffer from the kit to sample, and the sample inverted five to ten times before resting at room temperature for five minutes. The solution was put into a column and centrifuged, followed by two 500 ul ethanol washes. A volume of 20 ul of molecular water was used to elute the sample, and they were tested on the Nanodrop One for cleanliness and the Qbit fluorometer for concentration values. 

    Some samples were diluted with more molecular water to help adjust the cleanliness values to be more optimal, in which another 20 ul were added and thoroughly mixed to homogenize before measuring again with the same tools. All samples were stored at minus 80 degrees Celsius for later use. 

Results:

    Table 1 is the first set of species put through the kit, but one specie, D. pimensis, was not sufficient the first time around so it was tested how it would look if this cleaned sample was again put the through the kit, which can be seen in the second set in Table 2. 

Specie	Concentration ng/ul	260/280	260/230
Pimensis	32.7	1.89	1.68
Xinjiangensis	202	1.88	1.51

 

Specie (post dilution)	Concentration ng/ul	260/280	260/230
Pimensis	14	1.85	1.69
Xinjiangensis	100	1.88	2.12

 

Specie	Concentration ng/ul	260/280	260/230
Pimensis	14.5	1.89	1.07
Aquatilus	18.6	1.89	1.17
Dajeoenenis	93.1	1.91	1.96

Discussion:

    This week was pretty worrisome for a few reasons. Not only is the likelihood of species dropping out and having to be redone much higher than initially believed, but the kit is not seeming to do a very reliable job of cleaning the DNA, as seen in some of the samples. It appears that if the DNA concentration is not significant, at least 50 ng/ul, the 260/230 will not be ideal. This is strange because it seemed as though this kit was exceptional at cleaning the DNA when it came to the experiments with D. sonorensis, but now it seems to work much less effectively. The prior experiments were done with an older version of the kit, so that now it has been since updated and the old version unavailable, and there is a possibility that the new version is less useful. Either way, there is little other option for the next steps so we will likely continue doing these cleanups and just redoing these samples as needed. Since direct alcohol precipitations haven't worked in the past either, this may not be a very good option but our best one.