The major, reoccurring problem in this project has been the ethanol precipitation of the RNA eluant from the PAGE gel. This issue, along with problem solving techniques used and changes made in each round, is discussed at length in the previous section and in the first progress report. Also, the UV light camera used to photograph agarose gels wasn’t working properly during round two. This is why the round 2 cycle course gel picture (Fig 1) is blurry. The camera and software used to analyze the image were fiddled around with until they finally worked. The 2X blue dye used to visual the RNA crashed out of solution. It is now being kept at room temperature instead of the freezer to prevent this.
Conclusion and Future Work
The final concentrations of RNA at the end of each round were 989.8 and 1124.7 ng/ul for rounds 2 and 3 respectively (Fig 3). Ten cycles of PCR were used for round 2, and 9 cycles were used for round 3. To prevent future ethanol precipitation problems, the PAGE gel chunk will be eluted with 1ml instead of 3ml. Round 4 is already under way and the results will be included in the final manuscript. By that time, I plan on completing at least one or two more rounds and possibly a binding assay.
Round Large Scale PCR Cycles Absorbance Quantity of NA Used Quantity of NA Recovered
1 20 16.245 400 pmol 41988 ng
2 10 11.962 400 pmol 29694 ng
3 9 13.031 400 pmol 33741 ng
4 20 Not completed 400 pmol Not completed
Figure 3. Data from each round of selection for asynuc from the N34 pool.