IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-4

PAGE of Concentrated Eb, Fb, and Gb

 * given amounts of streptavidin were added to each component on parafilm
 * 2uL of 10x loading dye was added to each mixture subsequently
 * gel was run at 4&deg;C for 1hr 10minutes at 100V
 * gel was stained in coomassie blue from 12PM F 8/4/06 to 3:30PM Sa 8/5/06

PEG precipitation w/ New Folded Rxns

 * Goal: test PEG precipitation with folding rxns under new conditions
 * Plan to use 50 final volume
 * Add 10 10 nM scaffold/100 nM oligo folded mix (since there was only 20  in total of each folded rxn)
 * Add 20% PEG/2.5M NaCl solution (10 )
 * Add as much water to each as it takes to get them to a 100 final volume (30 )
 * Incubate on ice for 15 minutes
 * Spin 16k rcf for 10 minutes
 * Pipette out supernatant into separate tube
 * Resuspend pellet in 1x folding buffer volume equal to the supernatant
 * Run equivalent volumes of pellet and sup on 2% agarose gel (+ 11 mM )


 * Gel Analysis
 * Analyze on 2% Mg agarose gel (0.5x TBE, 11 mM MgCl2, 0.5 μg/mL EtBr)
 * Load 1kb ladder (1 lane)
 * Load naked scaffold (1 lane)
 * Load 6 folding rxns untreated, supernatant, pellet (18 lanes)
 * 20 lanes total


 * Results
 * no bands at all besides naked scaffold
 * have to redo 6 folding rxns

3D Design
Began working on 3D Design 5 schematic in Google Sketchup. Preliminary look below. CG equivalents of EM images posted next to EM images. Scales to come.