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

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** attachment DNA used: [[IGEM:Harvard/2006/Container_Design_4/Oligos#Attachment_Oligos_.28Ordered_8.2.29|c4.0.6.1ob]] (37 bp)
** attachment DNA used: [[IGEM:Harvard/2006/Container_Design_4/Oligos#Attachment_Oligos_.28Ordered_8.2.29|c4.0.6.1ob]] (37 bp)
** oligo-ligand: [[IGEM:Harvard/2006/DNA_nanostructures/Notebook/2006-7-31#Random_generation_script|script output]] (45 bp)
** oligo-ligand: [[IGEM:Harvard/2006/DNA_nanostructures/Notebook/2006-7-31#Random_generation_script|script output]] (45 bp)
-
* incubated at 37{{c}} for 40 min.
+
* incubated at 37{{c}} for 60 min.
-
* 2 {{ul}} loading dye added
+
* 10 {{ul}} each reaction (with 2 {{ul}} loading dye added) run on 18% Tris-Gly PA gel for 100 min. at 120 V, stained with SYBR Gold, imaged under EtBr filter
-
* 20 {{ul}} each reaction run on 18% Tris-Gly PA gel for 1.5 h at 120 V, stained with SYBR Gold, imaged under EtBr filter
+
** other 10 {{ul}} stored at 4{{c}} in case another gel needs to be run
 +
 
 +
[[Image:20060816_proassay.jpg|thumb|18% Tris-gly PAGE]]
{| {{table}}
{| {{table}}
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| 10||-ligand||10 {{ul}} 10 nM 8%-PEG-purified, ligand-incubated c5.0.A||2 {{ul}}||2 {{ul}}||1 {{ul}} 500 U/mL||5 {{ul}}
| 10||-ligand||10 {{ul}} 10 nM 8%-PEG-purified, ligand-incubated c5.0.A||2 {{ul}}||2 {{ul}}||1 {{ul}} 500 U/mL||5 {{ul}}
|}
|}
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==Magnetic Streptavidin Protection, Take 2==
==Magnetic Streptavidin Protection, Take 2==

Revision as of 17:57, 16 August 2006

Contents

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


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

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 60 min.
  • 10 μL each reaction (with 2 μL loading dye added) run on 18% Tris-Gly PA gel for 100 min. at 120 V, stained with SYBR Gold, imaged under EtBr filter
    • other 10 μL stored at 4[[:Category:{{{1}}}|{{{1}}}]] in case another gel needs to be run
18% Tris-gly PAGE
18% Tris-gly PAGE
lane trial DNA 10x NEBuffer 4 10x BSA AscI water
110 bp+ ladder5 μL
2-nanostructures -ligand1 μL 1 μM attachment DNA2 μL2 μL1 μL 500 U/mL14 μL
3-nanostructures -attachment1 μL 1 μM oligo-ligand2 μL2 μL1 μL 500 U/mL14 μL
4-nanostructures -enzyme1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand2 μL2 μL0 μL14 μL
5-nanostructures1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand2 μL2 μL1 μL 500 U/mL13 μL
6outward-facing ligands10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.C2 μL2 μL1 μL 500 U/mL5 μL
7inward-facing ligands10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.D2 μL2 μL1 μL 500 U/mL5 μL
8-oligos2.25 μL 44 nM p73082 μL2 μL1 μL 500 U/mL13.75 μL
9-enzyme10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.C2 μL2 μL0 μL6 μL
10-ligand10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.A2 μL2 μL1 μL 500 U/mL5 μL

Magnetic Streptavidin Protection, Take 2

  • Mistakes:
  1. The Speedvac was allowed to proceed for an hour. Upon return, what had been thought to be 200uL of final-supernatant had completely dried - unless the tubes that had been speedvaced were not in fact the tubes that had contained the final-supernatant, but intermediate tubes. Because the initial final-supernatant had appeared slightly brown, these tubes were placed back into the MagnaRack and the supernatants were re-extracted into different, final tubes. These tubes might have accidentally been thrown out in the cleaning of the lab area that occured while I was out to lunch. If so, there might be some small residual amount of elute which might show up in the gels, but is unlikely to. If this is the case, the experiment will need to be repeated (cost of beads: ~$32).
  2. If the tubes were the correct ones, the fact that they were Speedvaced to drying might have damaged the nanostructures irreparably and made them difficult, if not impossible to visualize in the gel. But hopefully scaffold and oligos will still be able to be seen.
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