Progress Report Two for Selection Against MUC-1

The original abstract can be found here that may assist in reacquainting the reader with the target. The proposal can be found here.

Conditions

Target: MUC1 protein His Tag

Beads: Nickle-NTA beads

Pool: N58

Incubation Time and Temperature: 37 degrees C for 45 min

Buffer: PBS 10X Selection Buffer

Wash volume and number: 3 washes, 2 volumes

Progress, Results and Discussion:

Two rounds of bead-based selection have been completed with minimal progress and round three is currently being done. Round 2 started with an initial concentration from the RNA in Round 1 to be 902.52 ng/uL. First a new stock of 10X PBS Selection Buffer was made using PBS and the MgCl2 salts, then the 10X PBS Selection Buffer was diluted to make 1X Selection Buffer for the Round. Next the target was immobilized on the Ni-NTA beads. A pool binding reaction was made with the PBS that was made today and the Binding and Selection took place with a total number of three washes. After eluting the RNA, and ethanol precipitation was done, reverse transcription to convert the RNA into DNA. Then a Cycle Course was done in order to recognize what cycle to run the Large Scale to. After the Large Scale was run to the approximate number of cycles, another ethanol precipitation was performed, followed by transcription, PAGE and elution.

Problems:

Round two faced many problems starting with reverse transcription so the first cycle course failed as seen by Figure 1. The results seemed unusual because the dye seemed to have run at an equal rate for all the E1 cycles and didn’t show distinct bands, except for wash 0. Another cycle course was repeated and nothing amplified again as seen by Figure 2, so reverse transcription was repeated. Finally, after the third cycle course of this round, the results in Figure 3 were shown. Although Cycles 12 and 15 in W0, and Cycles 15 and 20 in E1 seemed to be slightly over amplified, Cycle 15 was chosen for the large scale PCR since it had the overall best banding. After large scale was performed and an ethanol precipitation, transcription was done with Kit O and a PAGE gel was set up. However nothing showed up during the first PAGE, the gel didn’t set completely in the second PAGE and so another PAGE was set up. This time the gel didn’t polymerize and so another PAGE was set up, on this PAGE the gel polymerized everywhere except around the wells. Finally Travis set up a gel and I added my two samples in with it. Unfortunately nothing amplified; most likely due to overexposure to DNase (the enzyme cut up the DNA during transcription since there seemed to be fragments (a shadow) after the dye stopped). Transcription was repeated with Kit M and a PAGE gel was run on Sagar and Michael’s PAGE that they had set up. After eluting and precipitating the RNA a 1126.9ng/ul concentration was found.

Conclusions and Future Work

The final concentration of the MUC-1 in the N58 pool after Round 2 was 1126.9ng/ul. Cycle 15 was chosen for large scale after three cycle courses. In the future, a transcription will be done quicker, more efficiently, and with newer, practiced kits. Also a PAGE gel will be set up more efficiently, preferably with other people. Round 3 is nearly done and is expected to be available for the final paper.

Figure 1: Round 2 Cycle Course MUC-1: A 3.8% agarose gel was run in order to decide what cycle to run the large scale PCR to, unfortunately it failed and so another gel was run.

Figure 2: Round 2 Cycle Course Try 2 MUC-1: Another cycle course was performed and an agarose gel was run with the new RNA, however a new reverse transcription was not performed before the cycle course. There was absolutely no banding, and so reverse transcription was repeated.

Figure 3: Round 2 Cycle Course Try 3 MUC-1: After the second reverse transcription of this round, the final cycle course was performed and the above results were shown. The best banding was around cycle 15, which was chosen for the number of cycles for large scale. There were bubbles in some of the wells on the second row and so they were skipped.

Current Progress for MUC1 aptamer Bead Based Selection with N58 Pool
Round	LsPCR Cycles	Quantity of NA used	Wash # and volume	Concentration (recovered in uM)	Concentration (recovered in uM)
1	12	800pmol	3w/2v	902.52 ng/uL	32.7 uM
2	15	400pmol	3w/2v	1126.9 ng/uL	40.830 uM
3	Pending	400pmol	3w/2v	Pending	Pending

Table 1: Current Progress of 2 rounds of selection shown above.