Umar Ahmad - Progress Report 1 - FliC (Multimeric)

Progress Report 1: Bead-Based RNA Aptamer Selection Against the Mutlimeric Form of FliC

Thus far, 2 successful rounds of selection have happened and a third is in progress. This progress report will discuss in detail the progress made as well as problems encountered and the meaning of results obtained thus far. This report will follow a chronological order; first discussing the first round of selection, followed by the second and third, and lastly a general analysis of what has thus far been completed.

Round 1 was completed without many problems at all. Binding and selection as well
reverse transcription were completed without any difficulty. Next, cycle course PCR was performed. The following is the gel that was obtained:

Figure 1: Gel from Cycle Course PCR from Round 1

This gel shows a few interesting and notable things. First off, there is massive amplification after the 100 base pair point in all samples. This will be discussed in detail in a latter section of this report. It is also important to mention that a second ladder was mistakenly inserted into the gel without dye, thus it does not appear. Also, a no template PCR control was not run as it should have. In summary, this gel shows that the ideal number of amplification cycles was about 7 rounds of amplification. The rest of the round was completed without much difficulty. The following diagram shows an example representation of the PAGE run at the end of the round:

Figure 2: Representation of a PAGE. The Blue Area Signifies the RNA.

The quantitation indicated that the concentration of RNA in the band excised was 1011 ng/µl. This completed round 1 and thus round 2 was started.

Round 2 went similarly to round 1. As before, binding and selection as well as reverse transcription went without error. The following image is the gel from the cycle course from round 2:

Figure 3: Gel from Cycle Course PCR from Round 2

This gel is much better than the 1st as wash 3 amplified far later. The gel also shows that the ideal number of cycles for large scale PCR is about 10 cycles. The rest of the round including large scale PCR, transcription, and the PAGE were completed successfully. The concentration of RNA at the end of the round was 1132.9 ng/µl.

In the completion of these 2 rounds, some problems were encountered. The major problem was the appearance of items on the gel past the 100 base pair mark in every sample run. It was determined after discussion with others in the lab that these were just primers showing up. This isn’t of any concern, as it does not affect results. Aside from that, selection has been mostly successful. So far in round 3, binding and selection as well as reverse transcription have been completed.

As far as future work is concerned, selection will continue as described. The current goal is to complete 7 or 8 rounds and do a binding assay to determine binding affinity and proceed from there. The following table summarizes results thus far attained:

Table 1: Results from the 1st 2 Rounds of Selection against FliC using the N70 Pool

No comments: