IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-9

Folding reaction
Fold 200 each of 3.2.Fo, .Go, .Ho, .Io

Each reaction:
 * 45 p7308 scaffold (44 nM)
 * 80 oligo stock (250 nM)
 * 20 10x folding buffer (500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM )
 * 55 d

Folding conditions:
 * high-volume protocol: start at 80, decrement 0.5 every 1 min. for 120 cycles.

Gel analysis
 * see below

More Microcon trials
Another protocol, inspired by Millipore's Concentrating and Desalting DNA or RNA with Microcon or Centricon Centrifugal Filters:


 * 20 folded nanostructures (4.0) into a YM-50 Microcon tube
 * 480 water added
 * centrifuged at 14,000 g for 6 min.
 * flow-through discarded
 * centrifuged upside-down at 1,300 g for 3 min. to collect retentate
 * 53 collected

Gel analysis

 * ran half of retentate, along with half of starting nanostructure volume for comparison
 * also ran 5 of each nanostructure folded today to see if they folded properly




 * results/discussion
 * Microcon yield is reasonably good, but lots of oligos remain
 * in the future: go back to repeating wash four times for 6 min each time (the maximum number of washes, according to Microcon)
 * folding reactions appear to have folded properly

Microcon purification of yesterday's folding reactions

 * speedvac'd c3.2F, G, H, I, Eb, Fb flowthrough for 50 min. (vol. def much higher than 40 )
 * ran 2% agarose gel +
 * 3 flowthroughs is probably not enough - notice pretty clear oligo smears: should probably do 5/6
 * don't know if oligo-ligand and latches were added to rxns before microcon: repeat folding of c3.2F, G, H, I and retest