Pilin Protein N35 selection (PilA)
October 18, 2011
The abstract to this progress report can be found here
Prior to my real protein target selection, I went through to practice rounds. To get to the amplification process, I had to go through the selection process. I had to pre-wash 40ul of streptavidin magnetic beads to purify the settings for the introduction to the target protein Pilin (PilA). After adding the target to the beads, washing and incubating, I added 3uL of N35 RNA pool. I isolated the unbound pool, by removing the supernatant, using a magnet (to collect the beads on one side) and re-suspending into 200ul of PCR Selection buffer, into a tube and labeled it W0. I washed the pool mixed with the beads and target three times and removed the supernatant of each wash into a tube and labeled W1, W2, and W3 respectively. After obtaining the RNA, I had to precipitate it to isolate it. I proceeded using the third wash (W3), the unbound pool (W0), and the elution (E1). To get ready for ethanol precipitation, I decided to make all the volumes equal, so I added 300ul of diH2O to W0 and 200ul to W3, since the highest volume was in E1- 400ul. To make sure everything was working fine and well, I located a blue-colored pellet at the bottom of the three tubes after ethanol precipitation. I, then, re-suspended the pellet and dissolved it in 10ul of diH20 for reverse transcription.
To finalize the selection round, I had to obtain a DNA sequence to be amplified in PCR and run on agarose gel, because an RNA template would not work in PCR due to the lack of certain enzymes that would have to be present to amplify RNA. Saying so, I had to reverse the transcription process using an enzyme called SuperScript II Reverse Transcriptase and a reverse primer. Using the thermocycler to provide the required environment and temperature for the process to occur, I ran reverse transcription and saved my ssDNA in my freezer box to carry out cycle course PCR the following day.
I ran cycle course PCR in order to determine which cycle best amplified the DNA sequence of E1, which is where the target attached to the nucleic acid is. I had to stand by the thermocycler to pause and take samples over time. After I finished the cycle course PCR, I ran a 3.8% agarose gel electrophoresis to obtain my results. Figure 1(my real target proteins) and 2 (practice round from last spring’s protein), show and summarize my results:
Figure 1. After doing cycle course, a 3.8% agarose gel was run at 100 volts for 65 minutes to determine which cycle is suitable enough to amplify E1 in large scale.
Figure 2. This is the gel obtained after running a practice cycle course using saved protein from April of 2010.
As shown in Figure 1, Cycle 9 of E1 was the best amplified result I obtained and, thus, it was picked to be the number of cycles used in large scale PCR. Unfortunately and for some unknown reason, one of the ladders did not show up and the other ladder barely showed up. Likewise, Cycle 6 of W0 did not fully show up. The bands shown in Figure 1 are not as bright as the bands that showed up in the previous practice round as shown in Figure 2 above and that hinted that more washes are needed for this protein. Along with the washes and elution, a “no-template” control was run in a separate well to test for contamination in the primers and enzymes used to carry out PCR and the result was negative. The lesson learned from this cycle course is that more washes are needed for the protein that I am selecting against and that the usage of the wrong dye might have led to the strange results obtained. One the practice round gel (Figure 2) all the samples, especially E1, were over-amplified, indicating the usage of more cycles than needed. After the cycle course PCR, I ran a large scale PCR. The purpose behind a large scale PCR is to obtain enough DNA to carry out the transcription process. I used 6 PCR tubes for 6 different reactions to maximize the gain of RNA out of PCR. After obtaining enough DNA for transcription, I had to precipitate the DNA using EtOH. In order to do the ethanol precipitation, I had to combine the PCR reactions into two equal volumes, so that it would be easier for me to do. One tenth of the volume of the reaction had to be Sodium acetate, which helps remove salts and unwanted substances around the DNA. I added 3ul of glycogen to be able to see DNA as a pellet. The rest of the volume was 100% ethanol, which is 2.5X volumes (832.5 ul). The total volume of the reactions was 1165.5ul in each tube. After centrifuging and separating the supernatant from the pellet, DNA was precipitated and was ready to be transcribed.
For transcription, I had to gather transcription enzymes to convert DNA into RNA. This was rather difficult, since all the transcription kits in lab were limited and lacking some components. I, however, managed to collect all the required components and moved on. The process required incubation at 37 degrees Celsius for 2-3 hours and I stopped the round at that point.
The gel obtained from the practice round showed over-amplification of the protein washes and elution, which indicated that more PCR cycles were actually ran than needed. After running PAGE an amount of 60.01 ng/ul of RNA showed up on the Nanodrop, showing that a big loss of the protein occurred during the practice round. Moving onto the real target selection, the first round failed half-way through when I found out that I used N35 DNA instead of N35 RNA. Therefore, I had to stop and redo everything again. The second time of running the first round of my new protein, I ran into several problems. I accidentally used SELEX PCR buffer throughout the whole round, which might have led to the strange results that showed up on the gel (Figure 1). Also, while carrying out cycle course PCR, I added orange dye that lacked EtBr to all the tubes. However, after the 20th cycle, I added the right dye to all the tubes again, giving a total volume of 7ul in each tube. This might have also led to the strange outcomes observed. While running the gel for the real round, I added more buffer to the rig which exceeded the maximum-level line, resulting in the formation of bubbles that topped the gel while running. I was told, however, that excess of TBE buffer should not affect the results.
Conclusion and Future Work:
After going through SELEX and binding PilA, PCR was carried out and the results were embellishing and great. Using the suitable PCR cycle, a well-amplified DNA fragment was obtained and transcribed to get an RNA fragment. Through transcription, the RNA molecule was acquired. PAGE and Nanodrop will be carried out this week to get more information about the progression of this round. Being done with the first round successfully paves a road to another round; this is going to be carried out in the near future. The lessons learned during this round will help in getting better results in the following rounds. Multiple rounds would add to the precision and accuracy of the aptamer wanted to bind the PilA, which would help diagnose melioidosis and the presence of the Burkholderia protein.