Progress Report 1

Progress Report 1
Leta Ko
October 18, 2011
Fall 2011
Pool N58, Lysozyme

Progress, Results and Discussion
On September 1, selection against lysozyme began with target selection using biotinylated beads. The mix binding pool reaction was done with the N58 pool. The mix binding reaction was not done properly, thus the selection was restarted using the same beads. The beads were rewashed multiple times to ensure the N58 pool binding was all gone. Three washes were prepared for the cycle course PCR.  For the elution was, 2 volumes of 200ul was used. To have an equal amount throughout the washes in preparation of cycle course, an additional 200ul of diH20 was added to wash 1 and wash 3. A gel is performed with the cycle course PCR aliquot to reveal which cycle amplifies the DNA the most without over amplification or under amplification. Shown in Figure 1, the “no template control” was not performed along with cycle course; however the most optimal cycle was the 12th cycle.
For ethanol precipitation, the amount of sodium acetate was changed to 30ul to adjust the total volume of the large scale PCR. The two pellets of transcription were each resuspended with 20ul of diH20, instead of 10ul of diH20. The transcription master mix had a higher concentration of DTT, 10X TNX buffer, ATT, because of the addition of these components in the RNA stock resuspension pellet. On October 1st, PAGE was set up using 8% denaturing acrylamide. PAGE is performed to isolate the RNA from the excess unbounded RNA and other molecules from transcription. This RNA, that is concentrated with PAGE will provide as the RNA stock for the next round of selection. To this solution we added, 25ul of TEMED and 100ul of 10% APS. PAGE was performed, model shown in figure 2, with the RNA from transcription. TE buffer was made by Camille Ailean on October 1st. Ethanol precipitation was not performed on the proper day after the proper amount of incubation in 37°C, due to the painter lab being closed.  On October 3rd, ethanol precipitation could not be completely due to the lack of pellet that did not form during chilled centrifugation. The RNA solution was centrifuged for an hour, but there was no pellet found, thus no nano drop was performed.                  

Figure 1- Practice Round 1 cycle course gel: The most optimal band was shown in cycle 12 of the elution 1 wash. The quality of this gel picture is not as clear as it usually is, due to camera malfunction. The gel was left overnight in the freezer, and taken the next day.
Figure 2-Model of what was shown in the PAGE gel: The RNA was cut out and put into a conical tube.
On October 6th, another round of selection against lysozyme was started. Target immobilization was performed with newly made 1X PBS buffer and the mix pool binding reaction was done with the N58 pool. However, the first wash with PBS buffer was not saved. Because W0 was accidentally thrown away, W1 was used in cycle course PCR. Ethanol precipitation was performed and the pellet was resuspended with 10ul of diH20. Reverse transcription was set overnight and kept in the -20°C freezer for two days. On October 13th, cycle course PCR (shown in figure 3), W1, W3 and E1 was used along with a “no template control.” The gel provides a visual confirmation of which cycle would be the best amount of cycles to amplify the DNA. The gel was done with 100V, using 3.8% ethanol bromide; however the ethanol bromide rose toward the black electrode. This created a dark cloudy shadow on my gel, shown in Figure 3. However, there was no band on the “no template control.” The optimal cycle was the 15th cycle. Large scale PCR was performed with a new aliquots of 4mM DNTPs and ethanol precipitation was performed afterwards. Most of the supernatant was decanted and pipetted out, so the pellet only ran for 7 minutes in the speedvac. Each pellet was resuspended with 10 ul and transferred together into one tube. At the moment, transcription has finished and the RNA solution is prepped with DNAse and quenched it with 21ul of 2X blue denaturing dye.


Figure 3- Cycle Course Gel for Practice round 1: Negative template control shows a slight band, but could be from contamination from the overflow of the other wells beside it. Excess ETBr showed up as a cloud over my W1 bands. The top level 100 bp ladder is dim compared to the second level because of the volume of 100 bp ladder added into the wells of the gel.

Problems encountered
There were many problems encountered during the two practice rounds of selection against lysozyme. During the first target immobilization, I accidentally added the RNA (mixing pool reaction) to the beads. Therefore, I was not able to heat denature the mixture. Instead, I redid target immobilization using the same beads, after washing them repeatedly to rid of any bindings. Another error was made when we did cycle course PCR, we forgot to add on a “no template control” to ensure there is no bands shown when there is no DNA present. After large scale PCR and after ethanol precipitation, the DNA was resuspended with twice the volume. Instead we should have resuspended one of the pellet with 10ul and add the suspended DNA into the other pellet and add an additional 10 ul of diH20 to ensure that both pellets were resuspended and the total be kept at 20ul. Transcription was badly done due to the fact that I prepared the transcription master mix in the resuspended DNA. This was supposed to be done in a brand new tube. This error was only realized after DTT, 10X TNX buffer and ATT was added into the resuspended RNA. Instead of restarting over the entire round, we created a new master mix and added the regular amount of the recipe and still added 5 ul of the ds DNA.  During PAGE set up, we left the DNase and the 2X blue denaturing dye in the RNA PCR tube for too long. This was due to the fact that we were waiting for the PAGE gel to polymerize. These mistakes could have been the reasons why there was no pellet shown when we centrifuged the RNA after the PAGE gel and the incubation period. 

Round 1 for the selection against lysozyme was performed again to ensure that we knew how to perform each protocol perfectly. However, I forgot to save my W0 after washing the beads with PBS buffer. For the cycle course PCR, I did not aliquot 2ul of the DNA template, but instead added 10X PCR buffer, 4mM DNTPs, and reverse and forward primer into the “stock” of the DNA template. Instead of restarting over, I decided to take 2ul of the unfinished mixture and added the correct amount of components for PCR. I decided to use the unfinished mixture for large scale PCR. For my gel, the orange dye was running upwards to the positive end and created a gray cloud over my bands in the wash 1 section of my gel. Thankfully, I could still see the gel. However, next time, I will run my gel a bit longer to have the orange dye completely removed. 

Conclusion and Future Work
The purpose of the two practice rounds was to get familiarized with the steps of a round. This was incredibly beneficial for me, because of the immense amount of mistakes I performed. I will complete the last practice round with PAGE and begin my round for my target, myostatin. The two rounds took a longer time to complete due to the fact that I was mentoring a new member into the stream. It took us awhile, because of the mistakes I made, along with the responsibility to help the new member with her practice rounds and teaches her through each step of a selection. I plan on starting my actual target selection by next week and have 2 successful rounds of selection completed by the next progress report.

No comments: