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

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