IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-16

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Contents

Container v3.2

c3.2 Folding Experiment

Reagents (each expt in respective 0.2 mL PCR tube)

Experiment Scaffold Oligos Folding buffer dH2O total volume
B9 μL p730816 μL 3.2.B.core (250 nm)4 μL 10x11 μL40 μL
C9 μL p730816 μL 3.2.C.core (250 nm)4 μL 10x11 μL40 μL
6hb4.5 μL p73082 μL 6hb.v5 (0.99 μM)2 μL 10x11.5 μL20 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}}}]]

Latch-closing protocol

  • pipet 20 μL of expts B and C into respective clean 0.2 mL PCR tubes
  • add 1.0 μL of 3.2.B and 3.2.C (1.25 mM), respectively
  • incubate at room temp. for 30 min.

Gel analysis

  • 2% agarose gel supplemented to 10 mM MgCl2
    • 100 mL solution with 5 μL 10 mg/mL EtBr
  • run in 1x TBE supplemented to 10 mM MgCl2
  • 35 min at 130 V
Lane Contents Loading Buffer (10x TBE/glycerol)
11kb DNA ladder (10 μL)1.1 μL
2control: +scaffold -oligos (10 μL 23 μM)1.1 μL
3control: -scaffold +3.2.B.core oligos (10 μL 25 μM)1.1 μL
4expt B -latches (10 μL)1.1 μL
5expt B +latches (10 μL)1.1 μL
6control: -scaffold +3.2.C.core oligos (10 μL 25 μM)1.1 μL
7expt C -latches (10 μL)1.1 μL
8expt C +latches (10 μL)1.1 μL
9control: -scaffold +6hb.v5 oligos (10 μL 25 μM)1.1 μL
10expt 6hb (10 μL)1.1 μL
11control: +scaffold -oligos (10 μL 23 μM)1.1 μL
2% agarose gel electrophoresis
2% agarose gel electrophoresis

Results/discussion

  • expts B and C, with and without latches (lanes 4, 5, 7, 8, respectively), may have folded successfully, and run with approximately the same mobility as expt A
  • oligos appear very faintly in lanes 3-10 (appeared brighter before photograph was taken)
  • expts B and C without latches (lanes 4 and 7) appear to have similar motility to the same expts with latches (lanes 5 and 8, respectively), which is not conclusive as to whether the latch design was successful
  • 6-helix bundle (lane 10), as previously observed, has faster motility than p7308 scaffold (lanes 2 and 11)
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