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

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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°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
lane component concentration amount of component in well μL streptavidin added μL
1streptavidin2uM3.5-
2p730844uM7.53.5
3c4.0.6b (ie. biotinylated inside-oligos)50nM each oligo (as in all pre-working stocks)73.5
4Eb PEG-pellet21/8 of a rxn153.5
5Fb PEG-pellet21/8 of a rxn153.5
6Gb PEG-pellet21/8 of a rxn153.5
7streptavidin2uM7-

PEG precipitation w/ New Folded Rxns

  • Goal: test PEG precipitation with folding rxns under new conditions
  • Plan to use 50 μL final volume
  • Add 10 μL 10 nM scaffold/100 nM oligo folded mix (since there was only 20 μL in total of each folded rxn)
  • Add 20% PEG/2.5M NaCl solution (10 μL)
  • Add as much water to each as it takes to get them to a 100 μL final volume (30 μL)
  • 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 MgCl2)
  • 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


Lane Contents Loading Dye
01kb DNA ladder (4 μL)0 μL
1naked scaffold (10 μL)2 μL
210mM MgCl2 + 100nM oligos rxn untreated (10 μL)2 μL
310mM MgCl2 + 100nM oligos rxn pellet (10 μL)2 μL
410mM MgCl2 + 100nM oligos rxn supernatant (10 μL)2 μL
520mM MgCl2 + 100nM oligos rxn untreated (10 μL)2 μL
620mM MgCl2 + 100nM oligos rxn pellet (10 μL)2 μL
720mM MgCl2 + 100nM oligos rxn supernatant (10 μL)2 μL
830mM MgCl2 + 100nM oligos rxn untreated (10 μL)2 μL
930mM MgCl2 + 100nM oligos rxn pellet (10 μL)2 μL
1030mM MgCl2 + 100nM oligos rxn supernatant (10 μL)2 μL
1110mM MgCl2 + 600nM oligos rxn untreated (10 μL)2 μL
1210mM MgCl2 + 600nM oligos rxn pellet (10 μL)2 μL
1310mM MgCl2 + 600nM oligos rxn supernatant (10 μL)2 μL
1420mM MgCl2 + 600nM oligos rxn untreated (10 μL)2 μL
1520mM MgCl2 + 600nM oligos rxn pellet (10 μL)2 μL
1620mM MgCl2 + 600nM oligos rxn supernatant (10 μL)2 μL
1730mM MgCl2 + 600nM oligos rxn untreated (10 μL)2 μL
1830mM MgCl2 + 600nM oligos rxn pellet (10 μL)2 μL
1930mM MgCl2 + 600nM oligos rxn supernatant (10 μL)2 μL
  • 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.

Design 5

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