IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-21

Basic outline of the day

precipitate nanoboxes away from oligos (both 3.2 and 4.0) - using ethanol as we have no PEG — done
finishing folding 4.0 with latch 2 with biotinylated oligos for protection assay later
incubating biotinylated 4.0 structures (both precipitated and not) with streptavidin for imaging later
Tiff will come over to the med school for EM with 4.0 core, latch 1, latch 2, biotinylated core w/ strep, biotinylated core w/out strep, 6hb control
splitting up the scaffold based on chosen scaffold loop sites for design 5.0
designing latches for design 5.0
determining aptamer sites for design 5.0

Why precipitation? There are two problems with running a nanostructure and oligos together on a polyacrylamide gel with streptavidin. One is that it is unclear which band is which (see right), although we believe that the slowest band (just below the well) is the nanostructure and the faster smear is the oligos. The other is that the presence of excess (unfolded) biotinylated oligos will compete with the nanostructure for oligo binding. Therefore, we should precipitate our nanostructures in order to a) determine which band are the nanostructures and b) prevent biotinylated oligo interference.
Which precipitation protocol works best on our nanostructures? We should try all three of Shawn's protocols, and then run both the purified nanostructures, as well as the decanted supernatant, on an agarose gel to get a sense of purity/efficiency of each.

10 μL each of 4.0Db, 4.0Eb, 4.0Fb, 4.0Gb, and and 3.2.Ib were pipetted into respetive 0.2 mL PCR tubes precipitated with the salt and ethanol protocol.
The supernatant of each was reserved.
Each supernatant (Ib in lane 4, Db through Gb in lanes 5-8), the reconstituted Ib (post-precipitation) (lane 2), and unpurified Ib were run (lane 3) were run with loading dye (lane 1) and a 1 kb ladder (lane 9) on a 2% agarose gel.
- results: no data. gel box was turned around backwards, leading to everything running the wrong direction.
Had some difficulty during the reconstitution:
- reconstituted Ib with 10 μL 10x folding buffer (not 1x), Db and Eb with 5 μL 10x (not 1x), and Fb with 10 μL 10x (not 5 μL 1x). Gb was properly reconstituted with 5 μL 1x.

- Two tubes (770uL each) of pre-working stock 1 were made.
- We ran out of pre-working stocks 2 and 3 - whoever needs them next gets to make them, my carpal-tunnel's kicking in right now.
  - NB: It was noticed that, due to a typing error, previously the "total" for pre-working stocks 1, 2, and 3 were incorrectly listed as 77, 34, and 34 respectively. They are in fact 66, 33, and 33.
- Two tubes of folded Db and Eb were made yesterday; one of each were used in the precipitation today, along with the only tubes of folded Fb and Gb. In order to have enough rxns for the gel, another two tubes of Fb and Gb were folded along with the Hb and Ib created today.

Hb and Ib folding reactions were made up, along with additional Fb and Gb reactions.

goals:
- run purified DNA nanostructures on PA gel to determine its gel motility
- run scaffold and streptavidin together on PA gel to show that there is no scaffold-streptavidin interaction (we hope!)
- run concentrated, purified DNA nanostructures on a PA gel, with and without streptavidin, with biotin sites facing inwards and outwards, with and without lid latches that are added before streptavidin incubation
  - this will require us to choose the best precipitation protocol
method:
- run two identical 12% PA gel with all these combinations, including controls. stain one with Coomassie and the other with EtBr (which should image the double-stranded nanostructure strands just fine)
assumptions:
- the incubation conditions required for successful streptavidin-biotin are essentially that the two species need to be mixed at room temperature and that the required incubation time is no longer than a few minutes