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
1 streptavidin 2uM 3.5 -
2 p7308 44uM 7.5 3.5
3 c4.0.6b (ie. biotinylated inside-oligos) 50nM each oligo (as in all pre-working stocks) 7 3.5
4 Eb PEG-pellet 21/8 of a rxn 15 3.5
5 Fb PEG-pellet 21/8 of a rxn 15 3.5
6 Gb PEG-pellet 21/8 of a rxn 15 3.5
7 streptavidin 2uM 7 -

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
0 1kb DNA ladder (4 μL) 0 μL
1 naked scaffold (10 μL) 2 μL
2 10mM MgCl2 + 100nM oligos rxn untreated (10 μL) 2 μL
3 10mM MgCl2 + 100nM oligos rxn pellet (10 μL) 2 μL
4 10mM MgCl2 + 100nM oligos rxn supernatant (10 μL) 2 μL
5 20mM MgCl2 + 100nM oligos rxn untreated (10 μL) 2 μL
6 20mM MgCl2 + 100nM oligos rxn pellet (10 μL) 2 μL
7 20mM MgCl2 + 100nM oligos rxn supernatant (10 μL) 2 μL
8 30mM MgCl2 + 100nM oligos rxn untreated (10 μL) 2 μL
9 30mM MgCl2 + 100nM oligos rxn pellet (10 μL) 2 μL
10 30mM MgCl2 + 100nM oligos rxn supernatant (10 μL) 2 μL
11 10mM MgCl2 + 600nM oligos rxn untreated (10 μL) 2 μL
12 10mM MgCl2 + 600nM oligos rxn pellet (10 μL) 2 μL
13 10mM MgCl2 + 600nM oligos rxn supernatant (10 μL) 2 μL
14 20mM MgCl2 + 600nM oligos rxn untreated (10 μL) 2 μL
15 20mM MgCl2 + 600nM oligos rxn pellet (10 μL) 2 μL
16 20mM MgCl2 + 600nM oligos rxn supernatant (10 μL) 2 μL
17 30mM MgCl2 + 600nM oligos rxn untreated (10 μL) 2 μL
18 30mM MgCl2 + 600nM oligos rxn pellet (10 μL) 2 μL
19 30mM 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