Difference between revisions of "IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-16"

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(Protection assay)
(Repeat PEG Precipitation + Protection Assay)
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==Repeat PEG Precipitation + Protection Assay==
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==Repeat PEG Precipitation==
  
 
===Overview===
 
===Overview===
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* 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}}
 
* 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}}
  
 +
 +
[[Image:20060816_PEG.jpg|Gel]]
 
{| {{table}}
 
{| {{table}}
 
| align="center" style="background:#f0f0f0;"|'''lane'''
 
| align="center" style="background:#f0f0f0;"|'''lane'''

Revision as of 11:15, 16 August 2006

Repeat PEG Precipitation

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 μL 1x folding buffer with 30 mM MgCl2


Gel

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

Protection assay

  • used nanostructures folded above that were purified with 8% PEG, 0.5 M NaCl
  • mixed the following reactions, adding enzyme last
  • incubated at 37[[:Category:{{{1}}}|{{{1}}}]] for 40 min.
  • 2 μL loading dye added
  • 20 μL each reaction run on 18% Tris-Gly PA gel for 1.5 h at 120 V, stained with SYBR Gold, imaged under EtBr filter
trial DNA 10x NEBuffer 4 10x BSA AscI water
outward-facing ligands 10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.C 2 μL 2 μL 1 μL 500 U/mL 5 μL
inward-facing ligands 10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.D 2 μL 2 μL 1 μL 500 U/mL 5 μL
-oligos 2.25 μL 44 nM p7308 2 μL 2 μL 1 μL 500 U/mL 13.75 μL
-enzyme 10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.C 2 μL 2 μL 0 μL 6 μL
-ligand 10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.A 2 μL 2 μL 1 μL 500 U/mL 5 μL
-nanostructures 1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand 2 μL 2 μL 1 μL 500 U/mL 13 μL
-nanostructures -enzyme 1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand 2 μL 2 μL 0 μL 14 μL
-nanostructures -ligand 1 μL 1 μM attachment DNA 2 μL 2 μL 1 μL 500 U/mL 14 μL
-nanostructures -attachment 1 μL 1 μM oligo-ligand 2 μL 2 μL 1 μL 500 U/mL 14 μL