Selection Buffer: TRIS (10x diluted to 1x) pH 7.3
Incubation time/temperature: 45min/37*C
Washes/volume: 3 washes and 1 volume
Pool: RNA N34
Round 1 of selection against APP, pool N34, was started on the 20th of September 2010. A majority of the first week was spent Biotinlyating and re-suspending the target (in 1X PBS), after which the APP was aliquot into 20 tubes of 800pmol and 20 tubes of 400pmol. APP is a miniscule protein, 3717.1 kDal, which is why the two concentrations are necessary. R1 is being done using 400pmol, but in case the R1 binding assay doesn’t yield a high enough concentration, the round will have to be repeated using the 800pmol aliquot. This idea is based on the notion that a more concentrated target will result in a more concentrated binding assay.The rest of the time was spent on performing normal SELEX procedure. Cycle course PCR, however, had to be performed twice:
Figure 1: ccPCR, APP, N34, 9.28.10
As seen in Figure 1, the first attempt at PCR resulted in an overrun gel due to an overcharged and overexposed current, 115V for 35 minutes. The E1 samples ran over the W3 wells and resulted in an unreadable gel. The second run, performed under 90V for 25 minutes, provided much better results.
As seen in Figure 2, this PCR showed a good amount of amplification in the 9th cycle for E1. The over-amplified W3 was cause for some concern; it could be attributed to increased primer activity.
Large scale will be continued using 9 cycles, and a more concrete answer for the W3 over-amplification shall be determined by running a W3 cycle for 9 cycles. Running another gel, with this W3 sample, will determine if the primer is to blame for the results seen.
The first PCR agarose gel was overexposed, both in time and current, making the end result unreadable. This setback was expensive to the experiment in terms of time. Were it not for this mistake, two rounds of selection might have been possible by now. More care must be taken while determining gel conditions in the future. The second gel showed excessive amplification on the third wash. This could have been due to excessive primer activity, but the true root of this problem has yet to be determined; the end of this round should locate an answer.
Conclusion and Future Work:
This reporting period saw half of a round of selection; the lack of pace is mainly attributed to the mistake on the PCR gel. Although the pace is slow, there seems to be potential with target, especially with the amplification seen on the ninth cycle of E1 in Figure 2. With the selection moving forward, a cause for the W3 over-amplification will also be determined. Future work should also see the pace of selection pick up, leading to at least two more rounds and concrete binding assays (with concentration) by the next reporting period.