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

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(Repeat PEG Precipitation + Protection Assay)
(PEG ppt)
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===PEG ppt===
===PEG ppt===
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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}}
{| {{table}}
{| {{table}}

Revision as of 12:49, 16 August 2006

Contents

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 μL 1x folding buffer with 30 mM MgCl2
lane contents loading dye
18 μL untreated 5.0.A3 μL
220 μL 5.0.A in 7% PEG and 0.5 M NaCl (supernatant)3 μL
38 μL 5.0.A in 7% PEG and 0.5 M NaCl (pellet)3 μL
420 μL 5.0.A in 8% PEG and 0.5 M NaCl (supernatant)3 μL
58 μL 5.0.A in 8% PEG and 0.5 M NaCl (pellet)3 μL
620 μL 5.0.A in 9% PEG and 0.5 M NaCl (supernatant)3 μL
78 μL 5.0.A in 9% PEG and 0.5 M NaCl (pellet)3 μL
820 μL 5.0.A in 10% PEG and 0.5 M NaCl (supernatant)3 μL
98 μL 5.0.A in 10% PEG and 0.5 M NaCl (pellet)3 μL
101 kb+ ladder3 μL
112.7 μL p73083 μL
128 μL untreated 5.0.C3 μL
1320 μL 5.0.C in 7% PEG and 0.5 M NaCl (supernatant)3 μL
148 μL 5.0.C in 7% PEG and 0.5 M NaCl (pellet)3 μL
1520 μL 5.0.C in 8% PEG and 0.5 M NaCl (supernatant)3 μL
168 μL 5.0.C in 8% PEG and 0.5 M NaCl (pellet)3 μL
1720 μL 5.0.C in 9% PEG and 0.5 M NaCl (supernatant)3 μL
188 μL 5.0.C in 9% PEG and 0.5 M NaCl (pellet)3 μL
1920 μL 5.0.C in 10% PEG and 0.5 M NaCl (supernatant)3 μL
208 μL 5.0.C in 10% PEG and 0.5 M NaCl (pellet)3 μL
218 μL untreated 5.0.D3 μL
2220 μL 5.0.D in 7% PEG and 0.5 M NaCl (supernatant)3 μL
238 μL 5.0.D in 7% PEG and 0.5 M NaCl (pellet)3 μL
2420 μL 5.0.D in 8% PEG and 0.5 M NaCl (supernatant)3 μL
258 μL 5.0.D in 8% PEG and 0.5 M NaCl (pellet)3 μL
2620 μL 5.0.D in 9% PEG and 0.5 M NaCl (supernatant)3 μL
278 μL 5.0.D in 9% PEG and 0.5 M NaCl (pellet)3 μL
2820 μL 5.0.D in 10% PEG and 0.5 M NaCl (supernatant)3 μL
298 μL 5.0.D in 10% PEG and 0.5 M NaCl (pellet)3 μL
301 kb+ ladder3 μL
312.7 μL p73083 μL
32-40(empty)3 μL
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