Progress, Results, and Discussion
Two round of bead based selection with N34 RNA against s100A4 had been completed. Large scale PCR was performed at 12 cycles for R1 and transcription was performed. The PAGE after transcription showed a band that was unusually below the gel front and a crash soak was performed to determine RNA concentration. After transcription was determined to have failed, it was repeated, and successfully returned a significant concentration. Nanodrop spectrophotometry determined a concentration of 525.2 ng/uL which was calculated to be 19 uM. This was then used to determined that 5.26 uL of RNA would be used for round two.
Round 2 began with a negative selection to remove background binders and was continued with the same set of variables and conditions as the prior round. Cycle course PCR ran successfully showing increased concentration at lower cycles – possibly showing higher binding of target (Figure 1). Large scale was run at 8 cycles and transcription was ran but failed. Large scale was tested for binding and showed success (Figure 2). Transcription was repeated after twice and the third time was successful, but improper ethanol precipitation rendered it useless. The fourth time transcription failed again and the round maybe started over again completely.
Fig 1: ccPCR S100A4 R2. Successful amplification of
C8, C10, and C12. Optimal cycle seen as C8.
Fig 2: lsPCR S100A4 R2. Success in C8 lsPCR,
transcription was needed to be repeated
Many transcription failed due to various reasons. Some may have been bad kits, improper pipetting, or leaving the DNAse I in the reaction too long. The third transcription that worked, the gel was left in during ethanol precipitation rendering it useless after it was put in the -20C freezer. With great frustration with transcription, I may continue to repeat the entire round to have better success for future transcription.
Conclusion and Future Work
Round 2 will most likely be repeated all together, for better success in transcription.