IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-31

Folding & Analysis of design 5

• Folding
• Mix the following, for each of .1 to .5 (v1 and v2):
  • 4 uL 500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM MgCl2
  • 16 uL working stock 250 nM each oligo (100 nM each oligo final concentration)
  • 20 uL p7392 20 nM (10 nM final concentration)
• Anneal from 80°C to 20°C, -1°C per min

2% agarose gel, 0.5 mg/mL EtBr
0.5x TBE, 11 mM MgCl₂

Lane	Contents	Loading Buffer
0	1kb DNA ladder (4 μL)
1	naked p7308 (12.5 μL)	AGLB (4 μL)
2	c5.0 barrel (12.5 μL)	AGLB (4 μL)
3	naked p7704 (12.5 μL)	AGLB (4 μL)
4	c5.0 lids (12.5 μL)	AGLB (4 μL)

• Initial folding experiments of Design 5 barrel and lids
• Barrel
  • Used p7308 scaffold for barrel, as designed
  • Two bands are visible - one brighter band running at slightly faster mobility than naked scaffold, and a second more faint band running running slower, possibly indicating some dimization of the barrels.
• Lids
  • Used p7704 scaffold because p7572 isn't yet available (should be able to make some by end of Tuesday
  • significant amount of smearing is present, and high-molecular weight species are being retained at the top of the well
  • two slight bands are visible within the smear (again may be monomeric and dimeric species), hopefully indicating that some percentage is properly folded
  • will repeat with p7572 scaffold, possibly decrementing temperature -1°C per 2 min

Redesigning the DNA ligand

It turns out that NotI requires 10bp on each side of the restriction site for succesful digests. We need to redesign the old DNA ligand.

Other possible restriction enzymes (using NEB cleavage experiments to determine minimum number of bp needed on each side):

• AflIII (ACATGT)
  • sequence not present in p7308
  • >90% yield after 2 hr digest with 2 bp on each side
• AscIII (GGCGCGCC)
  • sequence not present in p7308
  • >90% yield after 2 hr digest with 0 bp on each side
• StuI (AGGCCT)
  • sequence not present in p7308
  • >90% yield after 2 hr digest with 1 bp on each side

None of these enzymes has confirmed star activity.

PEG precipitations

Used PEG precipitation protocol for reagents shown for lanes 5-20. Pellet lanes: reconstituted pellet in 10 μL folding buffer and loaded it all. Supernatant lanes: loaded 10 μL supernatant.

lane	folding reaction (μL)	20% peg / 2.5 M NaCl (μL)	water (μL)	final PEG concentration	pellet/supernatant
1	1 kb+ ladder
2	10	0	0	0%
3	10 (6hb)	20	10	10%	pellet
4	10 (6hb)	20	10	10%	supernatant
5	10	12	18	6%	pellet
6	10	12	18	6%	supernatant
7	10	16	14	8%	pellet
8	10	16	14	8%	supernatant
9	10	20	10	10%	pellet
10	10	20	10	10%	supernatant
11	10	22	8	11%	pellet
12	10	22	8	11%	supernatant
13	10	24	6	12%	pellet
14	10	24	6	12%	supernatant
15	10	26	4	13%	pellet
16	10	26	4	13%	supernatant
17	10	28	2	14%	pellet
18	10	28	2	14%	supernatant
19	10	30	0	15%	pellet
20	10	30	0	15%	supernatant

Gel purification of nanostructures

• using new buffer. added to the buffer 1 mL of 1M MgCl2 per 100 mL buffer
• using 20ul of folded structure.
• trying both design 3 and design 4
• using scaffold as a control (to hopefully varify we're cutting out nanostructures), but we know roughly where the nanostructures run to in any case.