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

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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
Igemharv06 2006-08-09 c3.2F.jpg
Lane Contents
1 10 μL nanostructures
2 26.5 μL retentate
3 1 kb+ ladder
4 p7308
5 5 μL 3.2.Fo
6 5 μL 3.2.Go
7 5 μL 3.2.Ho
8 5 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 EtBr
0.5x TBE, 11 mM MgCl2
Lane Contents Loading Buffer
0 1kb ladder (10 μL) -
1 naked p7308 (7.5 μL) 5 μL
2 c3.2F (7.5 μL) 5 μL
3 c3.2F flowthrough (35 μL) 5 μL
4 c3.2G (7.5 μL) 5 μL
5 c3.2G flowthrough (35 μL) 5 μL
6 c3.2H (7.5 μL) 5 μL
7 c3.2H flowthrough (35 μL) 5 μL
8 c3.2I (7.5 μL) 5 μL
9 c3.2I flowthrough (35 μL) 5 μL
10 c5.0Eb (7.5 μL) 5 μL
11 c5.0Eb flowthrough (35 μL) 5 μL
12 c5.0Fb (7.5 μL) 5 μL
13 c5.0Fb flowthrough (35 μL) 5 μL