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

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)