IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-11

Thoughts/ramblings/goals/questions/general frustrations
The results of yesterday's experiment show that Microcon filtration gives low yields and the PEG precipitation (at least at 10%) damages nanostructures regardless of folding conditions.

Questions
 * Are Microcon yields unacceptably low, or are they acceptable? (Can we use the NanoDrop to quantify our yield?)
 * Gels are much better for quantifying yield - you can try both and see how they compare.
 * Low concentrations of PEG should precipitate large nanostructures. Will some smaller concentration of PEG not harm nanostructures formed under some folding conditions?
 * Our August 2 experiment showed that even low concentrations of PEG damage nanostructures folded under "standard" conditions (10x oligos, 10 mM ).

Gigundo PEG precipitation

 * goal: test 0% to 8% PEG precipitations with nanostructures folded under all six folding conditions from yesterday
 * optimistic hypothesis: nanostructures folded with higher concentrations of oligos and/or will show less damage after treatment with PEG

Protocol: prepare the following 30 samples.




 * incubate on ice for 15 min.
 * spin at 16 k rcf at 4 for 10 min.
 * carefully pipet off supernatant
 * resuspend "pellet" in 20 of respective folding buffer
 * load resuspended pellets in odd-numbered lanes of 2% TBE agarose gel supplemented to 10 mM
 * load 30 (of 50 ) of supernatant into adjacent even-numbered lanes (e.g., trial 1-0 has pellet in lane 1 and supernatant in lane 2)
 * run at 60V for 1 h

Results/discussion
 * PEG precipitations appeared to have failed: no oligos were separated
 * curiously, the dye in the "supernatant" lanes ran at about 2/3 of the speed of the dye in the "pellet" lanes, and it gave a smear and not a band

Incubation of 3.2.E with thrombin beads

 * Goal: test if we can detect the binding of a nanostructure with outside aptamers to thrombin beads.

100 uL 10 nM 3.2.E or 100 uL 10 nM mix of 6.4. H/I was incubated with 250 uL thrombin beads (supplied as a 50% slurry). 3.2.E has outside aptamer sequences while 6.4.H/I do not. Following a 30 minute incubation, the beads were washed. The beads were then eluted by incubating with 250 uL 50% w/v free thrombin for 30 minutes. Washes and elutions were run on a 2% agarose gel for 60 minutes at 60V.