IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-14

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Container v3.2

Pre-working stocks

  • Mix 10 μL of each 50 μM stock oligo
pre-working stock desc oligos plate locations total
c3.2.1 core barrel 3.2p1A01–3.2p1H10 94
c3.2.2 core top lid 3.2p2A01–3.2p2C04 28
c3.2.3 core bottom lid 3.2p2C05–3.2p2E10 30
c3.2.4 barrel at inside apatamer locations -aptamers 3.2p2E11–3.2p2F01 3
c3.2.5 barrel at outside aptamer locations -aptamers 3.2p2F02–3.2p2F04 3
c3.2.6 barrel at inside apatamer locations +aptamers 3.2p2F05–3.2p2F07 3
c3.2.7 barrel at outside aptamer locations +aptamers 3.2p2F08–3.2p2F10 3
c3.2.8 barrel at latch locations -latches 3.2p2F11–3.2p2F12 2
c3.2.9 lid at latch locations -latches (empty) 0
c3.2.10 barrel at latch locations +latch1 +latch2 3.2p2G01–3.2p2G02 2
c3.2.11 latch from barrel +latch1 -latch2 3.2p2G03–3.2p2G04 2
c3.2.12 latch from barrel -latch1 +latch2 3.2p2G05–3.2p2G06 2
c3.2.13 lid at latch locations +latch1 +latch2 (empty) 0
c3.2.14 latch from lids +latch1 -latch2 3.2p2G07–3.2p2G08 2
c3.2.15 latch from lids -latch1 +latch2 3.2p2G09–3.2p2G10 2
c3.2.16 latch staples +latch2 3.2p2G11–3.2p2H02 4
c3.2.17 displacement strands latch1 3.2p2H03–3.2p2H06 4
c3.2.18 displacement strands latch2 3.2p2H07–3.2p2H10 4


Make c3.2 working stocks

Final concentration of each oligo in working stocks is 250 nM.

working stock description pre-working stocks (according to chart) water total
c3.2.A -latches
-aptamers
1 (94 μL), 2 (28 μL), 3 (30 μL), 4 (3 μL), 5 (3 μL), 8 (2 μL), 9 (0 μL) 40 μL 200 μL
c3.2.B.core +latch1
-aptamers
1 (94 μL), 2 (28 μL), 3 (30 μL), 4 (3 μL), 5 (3 μL), 10 (2 μL), 13 (0 μL) 40 μL 200 μL
c3.2.B.latches +latch1
-aptamers
11 (2 μL), 14 (2 μL) - -
c3.2.C.core +latch2
-aptamers
1 (94 μL), 2 (28 μL), 3 (30 μL), 4 (3 μL), 5 (3 μL), 10 (2 μL), 12 (2 μL), 13 (0 μL), 15 (2 μL) 36 μL 200 μL
c3.2.C.latches +latch2
-aptamers
16 (4 μL) - -

c3.2 Folding Experiment

Reagents

  • 9 μL p7308 scaffold = (10 nM)/(44 nM) * 40 μL
  • 16 μL oligos (3.2.A) = (100 nM)/(250 nM) * 40 μL
  • 4 μL 10x folding buffer (500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM MgCl2)
  • 11 μL dH2O
  • total volume: 40 μL

Annealing protocol

  • start at 80[[:Category:{{{1}}}|{{{1}}}]]
  • 60 cycles: wait 2 minutes, decrease 1[[:Category:{{{1}}}|{{{1}}}]]
  • hold at 4[[:Category:{{{1}}}|{{{1}}}]]

Gel analysis

  • 2% agarose gel supplemented to 10 mM MgCl2
  • run in 1x TBE supplemented to 10 mM MgCl2
  • 35 min at 130 V
  • 15 min in 1x TBE, 10 mM MgCl2, 100 μg/mL EtBr (forgot to put it in the gel)
Lane Contents Loading Buffer (10x TBE/glycerol)
1 1kb DNA ladder (10 μL) 1.1 μL
2 control: -scaffold +oligos (10 μL 25 μM) 1.1 μL
3 control: +scaffold -oligos (10 μL 23 μM) 1.1 μL
4 folded nanotubes (10 μL) 1.1 μL
2% agarose gel electrophoresis shows that the folding reaction (lane 4) contains a band slower than the DNA scaffold (lane 3)
  • results
    • ladder, scaffold, and oligos all appear as expected
    • folding reaction mixture shows oligo smear (expected) as well as a band that runs slower than the scaffold