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

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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
B 9 μL p7308 16 μL 3.2.B.core (250 nm) 4 μL 10x 11 μL 40 μL
C 9 μL p7308 16 μL 3.2.C.core (250 nm) 4 μL 10x 11 μL 40 μL
6hb 4.5 μL p7308 2 μL 6hb.v5 (0.99 μM) 2 μL 10x 11.5 μL 20 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)
1 1kb DNA ladder (10 μL) 1.1 μL
2 control: +scaffold -oligos (10 μL 23 μM) 1.1 μL
3 control: -scaffold +3.2.B.core oligos (10 μL 25 μM) 1.1 μL
4 expt B -latches (10 μL) 1.1 μL
5 expt B +latches (10 μL) 1.1 μL
6 control: -scaffold +3.2.C.core oligos (10 μL 25 μM) 1.1 μL
7 expt C -latches (10 μL) 1.1 μL
8 expt C +latches (10 μL) 1.1 μL
9 control: -scaffold +6hb.v5 oligos (10 μL 25 μM) 1.1 μL
10 expt 6hb (10 μL) 1.1 μL
11 control: +scaffold -oligos (10 μL 23 μM) 1.1 μL
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)