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 whether or not there are differences in molecular weight between methods, in comparison to each method’s difference in DNA yield after the initial lysis in the QIAGEN protocol.
Methods:
Deinococcus aquaticus was inoculated in five ml of TGY broth media and allowed 48 hours for growth. An OD 600 reading was taken and two tubes were made, each with one ml of solution and an OD 600 reading between 0.25 and 0.3. Four aliquots from each tube was made, and each was read for OD 600 reading, and then adjusted to a range between 0.27 and 0.35. All eight tubes were centrifuged for a cell pellet, supernatant removed, and resuspended in 250 micro liters of the Buffer B1 and Rnase solution according to the protocol. Each was vortexed on maximum speed for three seconds each. Five micro liters of lysozyme and 11.25 micro liters of proteinase K was added to tube 2B and incubated at 37 degrees Celsius, for one hour. RNA lysis buffer was added to tube 1A, the directions calling for 4 volumes to be added, which was a total of 1ml in this case. It was allowed to rest at room temperature overnight to be run on the gel the next day. All mechanical manipulation was conducted prior to incubation, after the addition of identical amounts of lysozyme and proteinase K to each tube. Incubation was done for 45 minutes at 37 degrees Celsius. Tube 2B was removed from incubation at 60 minutes, tested, and put back into incubation for an additional 30 minutes.
Tube Method (all bead beating done at 1m/s)
1A 1ml RNA lysis buffer
1B 30 second inversion
1C 4 second beat beating
1D 3 second bead beating
2A 45 second inversion
2B Lysozyme
2C 60 second inversion
2D 5 second bead beating
All samples were read on the Nanodrop 2000 and evaluated for ng/ul values. A 0.5% agarose gel was made identical to the protocol, and a HindIII ladder used as well. The samples underwent the cleaning process described, but another centrifugation step was added from last week. 175 micro liters of isopropanol was added to each, centrifuged, supernatant decanted, and 200 micro liters of ethanol used to wash each pellet. Another centrifuge was done and ethanol carefully removed. 20 micro liters of TE Buffer at pH 8.0 was used to resuspend each pellet. Ten micro liters of each sample was used in each well, along with two micro liters of UV dye. The gel was ran at 115 mA for approximately two hours.
Results:
The sample with RNA lysis buffer could not be tested on the Nanodrop, so no values were found for this sample. The rest of the tubes had initial readings shown in Figure 1 post lysis in the protocol. After the cleanup process described in the protocol, Figure 2 shows the readings done in TE Buffer per sample for the gel. The gel once again had no visible sign of bands or smearing. The ladder was prominent.
Tube Ng/ul 268/280 260/230
1A N/a N/a N/a
1B 77.6 0.72 -0.7
1C 81.8 0.62 0.26
1D 94.0 0.74 -0.76
2A 86.9 0.74 -0.75
2B
(60min) 135.0 0.7 -1.00
2C 81.3 0.71 -0.74
2D 95.1 0.76 -0.79
2B
(90min) 80.3 0.86 -1.82
Figure 1
Tube Ng/ul
1A N/a
1B 24.2
1C 16.9
1D 12.4
2A 28.2
2B
(90min) 22.6
2C 30.7
2D 26.3
Figure 2
Discussion:
The results are confusing to say the least. For one, there should have been some detection of DNA possible on the gel. Considering some wells had up to approximately 300 nanograms of DNA total, some evidence should have seen. The cleaning process seems to have incredibly significant loss, considering Figure 1 has a volume of 250 microliters and Figure 2 samples have a volume of 20 microliters. The total yield for these samples are in general shrinking from around 2 micrograms to a mere 400 nanograms, which is incredibly problematic. The readings from Figure 1 therefore may not be accurate although were previously thought to be. The plan going forward is to use a fluorometer that can measure the yield of DNA much more accurately than a Nanodrop can, as well as not needing a blank, which could be factoring into the results. We will also be using a separate DNA cleanup kit that contains a column procedure, to try and cut down on those losses. It is the goal to be able to load each well with at least 1 to 2 micrograms of DNA in order to get an accurate smear. The protocol also usually uses a different, much more sensitive, dye for their gels which could be the primary reason we have no seen any bands thus far. Further testing will be done to clarify these major questions, hopefully getting answers with more accurate testing with the fluorometer and being able to observe gel results using the separate DNA cleanup kit to prepare the samples.
Comments
Post a Comment