Progress Report two for Selection Against Amyloid Precursor Protein (APP)

Original abstract, useful in gaining an overview of the direction of the research, can be found here

Progress, Results and Discussion:
Two rounds of selection were completed using the bead-based selection protocol, using the following conditions
Target: APP
Beads used: Streptavidin
Pool: N34
Selection Buffer/PH: TRIS SELEX, pH 7.3
Ratio: 400 pmol APP: 200 pmol RNA
Incubation Time/Temperature: 20 minutes, at 37 *C
Wash volume/number: 1 volume (100ul), 3 washes.
The major concern for a majority of the experiment, and the main reason behind it falling so behind, was a series of failed transcriptions, using transcription kits N, M and A. After three failed PAGE runs, insufficient lsPCR product remained for another PAGE run, so it was necessary to restart the first round of selection. The cycle course images from both attempts at round one are presented below, in order, from least to most recent.

Figure 1: Cycle course PCR run from the first attempt at Round 1.

Figure 2: Cycle course PCR from the second attempt at Round 1.

As seen from FIGURE 2, the appropriate number of cycles, for the E1 wash, was just above the under amplified 15 cycle well, and so, 16 cycles were chosen. After the lsPCR of this second attempt, the product was run down another agaraose gel, simply to ensure that lsPCR had occurred successfully; as seen in Figure 3, below, there were no problems with the lsPCR.

Figure 3: lsPCR gel run to verify the success of large scale PCR.

Transcription finally worked, using kit B, and yielded a concentration of 404.4 ng/ul, which after all the failure, was quite encouraging. Round two of selection is already underway, and a PAGE should be completed early next week (targeting Monday), hopefully yielding another solid concentration.
Overall, the selection has been fairly hindered by the need to restart an entire round, particularly the first one. I would have liked to have a binding assay by the end of the semester, but seeing as the time constrain is fairly short, this goal will probably need some time input outside of the following week to be realised. I hope that I can at least get through 3 rounds of selection by the time the final report is due.

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