IFN-g Second Progress Report (Ashley Dawson)

The abstract and proposal can be found here.

Round One Conditions
Target: IFN-g with 6X His-tag
Beads: Nickel NTA
Pool: N34
Ratio of Pool to Target: 200:200 pmol
Buffer: 1X PBS with MgCl2
Incubation Time/Temp: 30 min at 37C
Wash Volume/Number: 3 washes, 2 volumes

After performing a large scale PCR for round one (see first progress report), the following PAGE purification did not show a band. One microliter of dsDNA from the large scale was run down an agarose gel with 5ul 6X Orange dye+ EtBr, and, as seen in Figure 1, no bands were present.


This implied that the large scale PCR had failed and round one was repeated with the same original conditions.

After repeating bead based selection for round one, another cycle course was performed and cycles six, twelve, fifteen and twenty were run down a 3.8% agarose gel as shown in Figure 2.

As cycle twelve had the best amplification, lsPCR was performed for twelve cycles. The large amounts of overamplification in W0 implies that most of the sequences are weak binders. This could either be attributed to low binding affinity of the target IFN-g or to too much stringency in the washes.

A PAGE was run to purify the round one RNA and the final concentration of RNA was 1911.9 ng/ul after elution and ethanol precipitation. This was more than enough move forward to round two.

Round Two Conditions
Target: IFN-g with 6X His-tag
Beads: Nickel NTA
Pool: N34
Ratio of Pool to Target: 200:200 pmol
Buffer: 1X PBS with MgCl2
Incubation Time/Temp: 25 min at 37C
Wash Volume/Number: 3 washes, 2 volumes

After completing another bead-based selection (with incubation time decreased by five minutes from the previous round) a cycle course PCR was performed on cycles seven, nine, thirteen and twenty. Only the elution was run fown the agarose gel as W0 and W3 were lost in ethanol precipitation. As shown in Figure 3, the elution amplified earlier than in round one implying that more strong binders were carried through the round.

A large scale PCR was run for nine cycles and, after transcription, a PAGE was run. After elution and ethanol precipitation the concentration of RNA was measured as more than 4000 ng/ul. As this was a greater value than the nanodrop's range of accuracy the RNA was diluted in an additional 30ul of 1X PBS SELEX and the new concentration was measured at 1315.5 ng/ul which was enough to move on to round three.

Round Three Conditions

Target: IFN-g with 6X His-tag
Beads: Nickel NTA
Pool: N34
Ratio of Pool to Target: 200:200 pmol
Buffer: 1X PBS with MgCl2
Incubation Time/Temp: 25 min at 37C
Negative Selection: 20 min at 37C
Wash Volume/Number: 3 washes, 2 volumes

In round three a negative selection was performed where an RNA pool binding reaction was incubated on only beads for twenty minutes. The supernantant from this was then incubated with the protein-bound beads. This was done in an effort to reduce the amount of background binders (sequences that bind to the beads or the tube).

After the negative selection, a cycle course was performed and cycles nine, twelve, fifteen and twenty were run down an agarose gel as seen in Figure 4.

The early amplification of W3 suggests that the wash process is not stringent enough as more sequences are still being washed away after two washes. This contradicts the previous data from round one which indicated that the wash proces was too stringent. This could be attributed to the possibility that a large number of the sequences in rounds one and two were binding to the beads or the tubes and when those sequences were removed there was a greater binding area for sequences that bound to the RNA. These sequences most likely included more weak and medium-strength binders than strong ones.

Large scale PCR was performed on cycle twelve and, following transcription, a PAGE was run. Due to bubbles in the gel, the RNA was pulled to the sides and so transcription was repeated and the PAGE run again. No bands showed up the second time either and so round three was restarted with the same original conditions. Another cycle course was performed, as seen in Figure 5, on cycles six, nine, twelve, fifteen and twenty of the elution and W0 only as W3 was lost in ethanol precipitation.

The early amplification was unexpected for the first round with negative selection especially as the previous round thre gel showed amplification at cycle twelve. This could be because of slight changes in the way the sequences stabilized after denaturation or differences in carrying out the protocol. However, a large scale PCR was run for six cycles as that was the optimum number indicated by the gel.

After transcription a PAGE was run and a very small band was shown. After elution and ethanol precipitation the concentration of RNA from the band was 141.8, too low to continue selection. Transcription and PAGE were repeated again and the band was even lighter. In an effort to get a higher yield, two transcriptions were then run and the bands from both combined. This gave a measured concentration of 205.4 ng/ul, still to low to continue. The failed PAGE gels are most likely either caused by something failing in transcription or large scale. If the PAGE fails again this round another test microliter of dsDNA will be run down an agarose gel to determine whether the problem is occurring during large scale.

Round three was restarted 11/29 and, after negative selection, a cycle course was run on W0, W3 and E3 for cycles six, nine, thirteen and sixteen as seen in Figure 6.

This indicates a relatively large amount strong binders as optimal amplification is around ten cycles. W0 amplified very early, as it has before in round three, which implies that the wash process is removing a lot of weak binders. The lack of amplification for W3 is similar to round one, indicating that the wash process removed all of the weak binders in the first two washes. A large scale PCR was run for ten cycles and round three will be continued and the concentration of RNA after PAGE elution will be measured to determine if selection can be continued.

The current progress and results are summarized in Figure 7.