Evaluating Methods for High Molecular DNA Extraction for Deinococcus aquaticus

 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 piece, for a total of eight testing samples. The readings of each tube was between 0.26 and 0.35. 

The QIAGEN protocol pictured below was followed for steps two through four. 250 microliters of B1 with RNase solution was used to resuspend the cell pellets, and five microliters of lysozyme was added to each tube (with the exception of Tube 2) and 11.25 microliters of proteinase K. All tubes were incubated for 45 minutes (with the exception of Tube 1). The mechanical step was added once all ingredients were added to each tube, but pre incubation. Samples were tested using a fluorometer with given standards, using five microliters of the sample. 

The second run of this experiment also followed the same protocol with a few changes. The first change was that the tubes were equilibrated to be within the 0.30 and 0.36 range for initial culture readings. Second, the mechanical step was done after the cells were resuspended in B1 and RNase solution, but prior to the addition of lysozyme and proteinase K. The samples were again tested on the fluorometer but ten microliters of sample was used for testing. 





Tube

Method

1

60 minute incubation period

2

Double lysozyme

3

30 seconds inverting

4

45 seconds inverting

5

60 seconds inverting

 

6

3 seconds bead beating

7

4 seconds bead beating

8

5 seconds bead beating
Figure 1 shows the tubes for the first trial this week. 

Tube

Method

1

15 seconds bead beating

2

30 seconds bead beating

3

45 seconds bead beating

4

60 seconds bead beating

5

Replicate 1

6

 

Replicate 2

7

Replicate 3

8

Replicate 4

Figure 2 shows the tubes done for the second trial of the week. 

Results:

    The results from Trial 1 show that most of the samples had less than what was recognizable to the fluorometer, which is approximately 0.17 ng/ul. The bead beating samples 6, and 7, had the highest values from the experiment. Trial 2 shows overall higher values than Trial 1, evident since each sample at least had a numerical value that could be determined. The most successful sample in the second run was Tube 2, although by a slim margin comparatively. 

Tube

Ng/ul

1

Too low (out of range)

2

0.42

3

Too low (out of range)

4

Too low (out of range)

5

Too low (out of range)

6

 

1.18

7

0.46

8

Too low (out of range)

Figure 3 shows the results from trial 1. 

Tube

Ng/ul

1

1.26

2

1.30

3

0.62

4

1.14

5

0.76

6

 

0.62

7

0.86

8

0.49

Figure 4 shows results from trial 2. 

Conclusions:

The results from this week leave more questions than answers. The nanodrop readings from previous tests indicated that the cells were being lysed, but the fluorometer is indicating otherwise. Due to the nature of the fluorometer and how it detects DNA, i tis possible such a dirty sample filled with all of the cell's components may be affecting the fluorometer's ability to effectively read how much DNA is actually in the sample. The nanodrop may not be entirely accurate, but at least indicated that cells have been lysed since something is being detected at all. Cleaning each sample will be necessary next week to properly test each one, to get a more definitive conclusion as to whether or not the cells are being lysed. If they are not, more mechanical manipulation is clearly needed, and if they are, then further testing needs to be done to determine how much there is exactly and at what molecular weight. There is a potential to use glass beads for more effective lysing if that is deemed necessary.

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