Progress, Results, and Discussion
The first round of aptamer selection for alpha-synuclein (asynuc) from the n34 RNA pool was completed on October 5, 2010. Despite a few minor setbacks, the first round showed promising results, and the second round of selection is already under way. Being a novice to filter selection, I expected to have some problems at first, but I’m confident that the subsequent rounds will go faster and have fewer problems.
Cycle course PCR was used in order to find the optimum number of cycles for large scale PCR. PCR product was taken out at the end of the certain cycles, and then the products were run down a 3.8% agarose gel to separate them, Figure 1. As you can see, not even the positive control, washes from round 1 (Wr1), showed up in the gel. This indicates that amplification of the DNA during PCR was unsuccessful. This might be due to bad PCR reagents.
To test whether or not the reagents used during cycle course PCR were working properly, a positive control with a DNA sample that had worked previously was performed. The control DNA, Wr1, and E1 were put through 9 cycles of PCR with the same reagents used in the previous cycle course PCR and then ran down a gel, Figure 2. The control DNA and Wr1 showed up, but E1 did not. This indicates the PCR reagents are working properly, but E1 hadn’t been amplified enough to show up in the gel yet.
Problems Encountered
Conclusion and Future Work
For the first round of RNA aptamer selection for asynuc, I got a final concentration of 1399.6 ng/ul of RNA from the n34 pool. I learned a lot about filter selection during this round and I also learned some problem solving techniques for cycle course PCR and ethanol precipitation. My first cycle course PCR yielded an unsuccessful gel. This problem was correct by repeating the cycle course after a control verified that the PCR reagents were working properly. By the next progress report I plan on completing at least 2-3 more rounds of selection.
The first round of aptamer selection for alpha-synuclein (asynuc) from the n34 RNA pool was completed on October 5, 2010. Despite a few minor setbacks, the first round showed promising results, and the second round of selection is already under way. Being a novice to filter selection, I expected to have some problems at first, but I’m confident that the subsequent rounds will go faster and have fewer problems.
Cycle course PCR was used in order to find the optimum number of cycles for large scale PCR. PCR product was taken out at the end of the certain cycles, and then the products were run down a 3.8% agarose gel to separate them, Figure 1. As you can see, not even the positive control, washes from round 1 (Wr1), showed up in the gel. This indicates that amplification of the DNA during PCR was unsuccessful. This might be due to bad PCR reagents.
To test whether or not the reagents used during cycle course PCR were working properly, a positive control with a DNA sample that had worked previously was performed. The control DNA, Wr1, and E1 were put through 9 cycles of PCR with the same reagents used in the previous cycle course PCR and then ran down a gel, Figure 2. The control DNA and Wr1 showed up, but E1 did not. This indicates the PCR reagents are working properly, but E1 hadn’t been amplified enough to show up in the gel yet.
Wr1 and E1 samples that had already been through 9 rounds of PCR were put through 11 more rounds of PCR, and samples were again taken during various cycles, Figure 3. This was done to see the optimum number of rounds for large scale PCR, and to make sure that something hadn’t gone terribly wrong with E1 in a previous step. As you can see, E1 only showed up on the 20th cycle of PCR, indicating that the product just hadn’t been amplified enough yet. Because of this, 20 cycles of PCR were used for large scale PCR.
Problems Encountered
The main problem faced in round 1 was the failed cycle course PCR, described above. Also, the lines in the agarose gels were all very faint so next time I will use more Ethidium bromide so they show up better under the UV light. I had trouble getting a pellet during the last ethanol precipitation as well. This is because I used an eluant with 3M NaOAc already in it to elute the RNA from the PAGE gel chunk, and then accidentally added more 3M NaOAc during the ethanol precipitation. This altered the delicate concentration ratios in the mixture and did not allow the RNA to precipitate. To fix this, I corrected the concentrations by adding 3ml of water and 8.25ml 100% ethanol. This time it worked, and a pellet was formed. For this ethanol precipitation I put the entire reaction in a 15ml conical. Next time I will divide the reaction up into 1.5ml tubes so that it will be easier to form a pellet.
Conclusion and Future Work
For the first round of RNA aptamer selection for asynuc, I got a final concentration of 1399.6 ng/ul of RNA from the n34 pool. I learned a lot about filter selection during this round and I also learned some problem solving techniques for cycle course PCR and ethanol precipitation. My first cycle course PCR yielded an unsuccessful gel. This problem was correct by repeating the cycle course after a control verified that the PCR reagents were working properly. By the next progress report I plan on completing at least 2-3 more rounds of selection.
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