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 |
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)