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Progress Report #2: RNA Selection for S100B
Progress, Results, and Discussion
Two rounds of selection for S100B using N34 have been completed. Both rounds were completed using a bead based selection protocol with PBS as the selection buffer, and a starting target ratio of 200pmol:100pmol. The binding reactions were washed, immobilized, suspended in 100ul of the pool binding reaction, and incubated at room temperature for 30 minutes.
Figure 1 below shows cycle course PCR for round 1. Cycle 10 showed the best amplification and was used to carry out large scale PCR. Transcription and a PAGE gel was performed and a band was seen, similar to figure 4. The RNA was eluted from the gel and the conical was placed in the 37C fridge overnight. An ethanol precipitation was performed and the RNA was quantitated. The resulting concentration, 27.1 ng/uL, was significantly lower than expected. This could be due to error during binding, buffer error, or a combination of multiple other factors. From this concentration, a calculated concentration of 0.98uM was utilized for round 2.
Figure 1: ccPCR_R1_S100B_
N34. Cycle 9 was slightly under amplified and cycle 12 was over amplified. Therefore, cycle 10 was chosen.
Negative selection was performed for round 2 to select for RNA that binds to the filter and not the protein. One set of beads contained only RNA while another set of bead contained the protein. Both sets were incubated and the supernatant from the tube without the protein was removed and added to the tube containing the protein. Selection, RNA precipitation and elution, reverse transcription, and cycle course PCR were performed. The resulting gel (Figure 2) showed that cycle 13 amplified the best and was used for large scale PCR. The protocol was followed for subsequent steps and a PAGE gel was performed. Trial 1 of the PAGE showed a small smear of a band and this was used to extract the RNA by crashing. After ethanol precipitation, the nanodrop revealed that the concentration, 4.4ng/uL, was too low to proceed further. Therefore, a 3.8% agarose gel was used to check for data in large scale PCR. As seen in figure 3, the gel revealed that data was present and another PAGE gel was run. A distinct band was seen as shown in figure 4. As in round 1, the protocol was followed for subsequent steps and the final RNA was quantitated. The concentration significantly increased to 184.9 ng/uL. The RNA was quantitated twice more to confirm the initial concentration. The second and third trials were about the same concentration, proving that 184.9 ng/uL was accurate. From this concentration, it was determined that 7uM will be used for round 3.
Figure 2: ccPCR_R2_S100B_N34. Cycle course during round 2 revealed that cycle 13 was appropriate for large scale PCR.
Figure 3: lsPCR_R2_S100B_N34. A 3.8% agarose gel was used to check for an adequate large scale PCR. A band was seen and a PAGE gel was run again.
Figure 4: PAGE gel seen after round 1 and round 2 for selection against S100B using the N34 pool. The RNA band was cut, suspended in 1X TE, and incubated overnight in the 37C fridge to extract the RNA.
Round 3 is currently in progress; selection has been completed.
Round 1 resulted in an abnormally low concentration of RNA (27.1 ng/uL). This could be due to multiple causes such errors during selection, thermocycler problems, or the protein itself.
Round 2 experienced problems during transcription and PAGE. During trail 1, transcription was performed using kits A and O and a PAGE gel was set up; however, a band was not seen. Transcription was repeated for trial 2 using kit B, a different transcription kit. The components were properly mixed and incubated overnight. A PAGE was poured and a smear of RNA was seen in the middle of the gel. This smear was cut out and the RNA was extracted via crashing. Quantitation revealed a low RNA concentration of 4.4ng/uL. This could be due to the use of the new transcription kit that had arrived earlier that week. These transcription kits had not been properly stored in the fridge; therefore transcription may not have been successful. Large scale PCR of round 2 was run in a 3.8% agarose gel to verify proper amplification. Figure 3 revealed that large scale was a success. Transcription was repeated using only kit O and a PAGE gel was run. A clear band was seen similar to figure 4. The RNA band was cut and eluted overnight as in previous rounds. Ethanol precipitation was performed but there were problems seeing a RNA pellet. Initially 3uL of gylcogen was added along with the other components, placed in the -80C fridge, and centrifuged. A pellet was not seen so this process was repeated for a total of three times. A pellet was still not seen so ethanol precipitation was repeated the following day and a pellet was seen. The RNA was quantified and the concentration was 184.9 ng/uL. As stated previously, this concentration was significantly higher than the previous round. The measurement was taken twice more to verify the accuracy and both measurements read at about the same concentration.
Conclusion and Future Work
Round 1 had a concentration of 27.1 ng/uL and round 2 had a concentration of 184.9 ng/uL. Cycle ten was used for round 1 and cycle thirteen was used in round 2 (Figures 2 and 3, respectively). To avoid future problems in subsequent rounds, transcription will be performed more carefully and with a transcription kit that is known to work from previous rounds. Round 3 is currently in progress and is expected to be finished for the final manuscript.
Figure 5: Data from each round of selection for S100B using the N34 pool.