IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-20

c4.0.b Folding

Pre-working stocks

• Mix 2.5 μL of each 200 μM stock oligo. 2.5μL biotinylated oligos + 7.5μL water go into each pre-working stock.

pre-working stock	desc	oligo tube labels	total # of oligos
c4.0.4b	barrel oligos @ outside aptamers -aptamers +biotin	c4.0.4.1b-c4.0.4.4b	4
c4.0.6b	barrel oligos @ inside aptamers -aptamers +biotin	c4.0.6.1b-c4.0.6.4b	4

Make c4.0.b working stocks

Final concentration of each oligo in working stocks is 250 nM.

Working Stocks for Biotinylated Oligos

Stock ID

Experiment

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

4b

6b

c4.0Db

no latches, inside biotin

x

x

x

x

x

x

x

x

c4.0Eb

no latches, outside biotin

x

x

x

x

x

x

x

x

c4.0Fb

latch design 1, inside biotin

x

x

x

x

x

x

x

x

c4.0Gb

latch design 1, outside biotin

x

x

x

x

x

x

x

x

c4.0Hb

latch design 2, inside biotin

x

x

x

x

x

x

x

x

x

x

x

c4.0Ib

latch design 2, outside biotin

x

x

x

x

x

x

x

x

x

x

x

c4.0.b 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

Streptavidin-biotin binding experiments, continued

• goal: show that streptavidin and biotin are binding and can be imaged with PAGE
  • show with both biotinylated oligos and biotinylated DNA nanostructures

lane	streptavidin (2 μM) (μL)	biotinylated oligo (1 μM) (μL)	water (μL)	loading buffer (10x Tris-glycine)
1	2	-	8	2
2	-	8	2	2
3	4	4	2	2
4	2	2	6	2
5	2	8	0	2
6	1	4	5	2
7-12	same as lanes 1-6

lane	streptavidin (2 μM) (μL)	box Ib (10 nM) (μL)	water (μL)	loading buffer (10x Tris-glycine)
1	2	-	8	2
2	-	4	6	2
3	2	2	6	2
4	1	1	8	2
5	1	4	5	2
6	0.5	2	7.5	2
7-12	same as lanes 1-6