IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-16

Repeat PEG Precipitation + Protection Assay

Overview

• concentrate on 30 mM MgCl₂ folding buffer, 1x oligos trial from yesterday (yielded best PEG results)
• folding rxns:

    32 (40 μL rxn) c5.0C (no latch, inside ligand)
    32 (40 μL rxn) c5.0D (no latch, outside ligand)
    8 (40 μL rxn) c5.0A (no latch, no ligand)

• repeat 8%, 10% PEG precipitation

Folding Reactions

• reconstituted the speedvaced 5.0.C and 5.0.D reactions from yesterday in 200 μL. Essentially we could have skipped the speedvac because as it turns out we're just going to be using the 30mM MgCl2. If we'd needed 20mM we would have reconstitued in 16 μL.
• Made more 10x folding buffer (300 mM MgCl2)
• folding rxns:

    32 (40 μL rxn) c5.0C (no latch, inside ligand)
    32 (40 μL rxn) c5.0D (no latch, outside ligand)
    8 (40 μL rxn) c5.0A (no latch, no ligand)

• Each rxn:

    16 ul oligos
    9 ul p7308
    11 ul H20
    4 ul 10x folding buffer

PEG ppt

• protocol: mix all ingredients, add nanostructures last, total volume is 100 mL, incubate on ice for 15 min, spin for 10 min at 16k rcf, pipet off supernatant, resuspend pellet in 30 {{ul)) 1x folding buffer with 30 mM MgCl₂

lane	contents	loading dye
1	8 μL untreated 5.0.A	3 μL
2	20 μL 5.0.A in 7% PEG and 0.5 M NaCl (supernatant)	3 μL
3	8 μL 5.0.A in 7% PEG and 0.5 M NaCl (pellet)	3 μL
4	20 μL 5.0.A in 8% PEG and 0.5 M NaCl (supernatant)	3 μL
5	8 μL 5.0.A in 8% PEG and 0.5 M NaCl (pellet)	3 μL
6	20 μL 5.0.A in 9% PEG and 0.5 M NaCl (supernatant)	3 μL
7	8 μL 5.0.A in 9% PEG and 0.5 M NaCl (pellet)	3 μL
8	20 μL 5.0.A in 10% PEG and 0.5 M NaCl (supernatant)	3 μL
9	8 μL 5.0.A in 10% PEG and 0.5 M NaCl (pellet)	3 μL
10	1 kb+ ladder	3 μL
11	2.7 μL p7308	3 μL
12	8 μL untreated 5.0.C	3 μL
13	20 μL 5.0.C in 7% PEG and 0.5 M NaCl (supernatant)	3 μL
14	8 μL 5.0.C in 7% PEG and 0.5 M NaCl (pellet)	3 μL
15	20 μL 5.0.C in 8% PEG and 0.5 M NaCl (supernatant)	3 μL
16	8 μL 5.0.C in 8% PEG and 0.5 M NaCl (pellet)	3 μL
17	20 μL 5.0.C in 9% PEG and 0.5 M NaCl (supernatant)	3 μL
18	8 μL 5.0.C in 9% PEG and 0.5 M NaCl (pellet)	3 μL
19	20 μL 5.0.C in 10% PEG and 0.5 M NaCl (supernatant)	3 μL
20	8 μL 5.0.C in 10% PEG and 0.5 M NaCl (pellet)	3 μL
21	8 μL untreated 5.0.D	3 μL
22	20 μL 5.0.D in 7% PEG and 0.5 M NaCl (supernatant)	3 μL
23	8 μL 5.0.D in 7% PEG and 0.5 M NaCl (pellet)	3 μL
24	20 μL 5.0.D in 8% PEG and 0.5 M NaCl (supernatant)	3 μL
25	8 μL 5.0.D in 8% PEG and 0.5 M NaCl (pellet)	3 μL
26	20 μL 5.0.D in 9% PEG and 0.5 M NaCl (supernatant)	3 μL
27	8 μL 5.0.D in 9% PEG and 0.5 M NaCl (pellet)	3 μL
28	20 μL 5.0.D in 10% PEG and 0.5 M NaCl (supernatant)	3 μL
29	8 μL 5.0.D in 10% PEG and 0.5 M NaCl (pellet)	3 μL
30	1 kb+ ladder	3 μL
31	2.7 μL p7308	3 μL
32-40	(empty)	3 μL