Progress Report 2-1027

The second cloning event was checked with a 3.8% agarose gel that failed, as shown below. Due to the base pair ladder not showing up, it was deemed an etBr failure. During this time, a third cloning event had already been started and grown up on a plate (per TA cloning instructions) so the second cloning event was scrapped and the third cloning event was then run on a gel.

This is the third cloning event plate. There are good white colonies that are significantly larger than the surrounding colonies.

40 Colonies were picked and then run down a gel to ascertain the insertion of plasmids:

Clones 1, 2, 8, 9, 11, 14, 15, 16, 18, 24, 25, 26, 28, 29, 30, 32, 33, 34, 36, 37, 38, 39 were sequenced

Clones 3, 19, 35, 40 were good to be sequenced but were not sequenced due to space issues

Clones 5, 6, 7, 10, 12, 13, 20, 21, 22, 23, 27, 31 were not sequenced.

Out of all of the sequences, clones 7, 11, and 14 worked with 11 and 14 being identical.

These will later be synthesized by an outside source and purified. After that, a binding assay will be run.

The sequence from clone plate 1:

This sequence was synthesized and a stemloop description was generated:

They generally look like aptamer structures so it is a positive step in the direction that we want for finding an binding aptamer. Once the sequence was in, it was PAGE purified, eluted, and run 6 cycles of large scale PCR to make the single strand double stranded.

The sequence from Clone plate 3 looks like this:

While these do not look like usual binding aptamers, there is a chance that it could be a binder, so a binding assay will be done anyways.

The binding assay for R7Bc71027 was performed once with a filter and once with beads, both leading to ambiguous results:

The graph above shows the filter binding assay which Holli performed. The first exposure of the cassette showed more binding than the second exposure (shown above) but it was proportionally about the same in regards to the background. Three reactions performed during the binding assay yielded three different percentages for binding and background binding, leading to the question of perhaps there was something that had gone awry during the exposure. The negative of the cassette showed a smear over one of the dots where the radiation had gone, perhaps due to a leaky gasket. Why there is background binding in a single sequence binding assay is questionable, which is why it was repeated as a Bead Binding Assay which is not pictured right now but there was an odd result with the W0, therefore Brad is currently repeating it.

Future work:

More binding assay work and possibly more cloning and sequencing to find a good binder.