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

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Contents

Folding reaction

Fold 200 μL each of 3.2.Fo, .Go, .Ho, .Io

Each reaction:

  • 45 μL p7308 scaffold (44 nM)
  • 80 μL oligo stock (250 nM)
  • 20 μL 10x folding buffer (500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM MgCl2)
  • 55 μL dH2O

Folding conditions:

  • high-volume protocol: start at 80[[:Category:{{{1}}}|{{{1}}}]], decrement 0.5[[:Category:{{{1}}}|{{{1}}}]] every 1 min. for 120 cycles.

Gel analysis

More Microcon trials

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

  • 20 μL folded nanostructures (4.0) into a YM-50 Microcon tube
  • 480 μL 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 μL collected

Gel analysis

  • ran half of retentate, along with half of starting nanostructure volume for comparison
  • also ran 5 μL of each nanostructure folded today to see if they folded properly
Lane Contents
110 μL nanostructures
226.5 μL retentate
31 kb+ ladder
4p7308
55 μL 3.2.Fo
65 μL 3.2.Go
75 μL 3.2.Ho
85 μL 3.2.Io
  • 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 μL)
  • ran 2% agarose gel + MgCl2
  • 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
2% agarose gel, 0.5 mg/mL EtBr0.5x TBE, 11 mM MgCl2
2% agarose gel, 0.5 mg/mL EtBr
0.5x TBE, 11 mM MgCl2
Lane Contents Loading Buffer
01kb ladder (10 μL)-
1naked p7308 (7.5 μL)5 μL
2c3.2F (7.5 μL)5 μL
3c3.2F flowthrough (35 μL)5 μL
4c3.2G (7.5 μL)5 μL
5c3.2G flowthrough (35 μL)5 μL
6c3.2H (7.5 μL)5 μL
7c3.2H flowthrough (35 μL)5 μL
8c3.2I (7.5 μL)5 μL
9c3.2I flowthrough (35 μL)5 μL
10c5.0Eb (7.5 μL)5 μL
11c5.0Eb flowthrough (35 μL)5 μL
12c5.0Fb (7.5 μL)5 μL
13c5.0Fb flowthrough (35 μL)5 μL
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