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

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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
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 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
lane trial DNA 10x NEBuffer 4 10x BSA AscI water
1 10 bp+ ladder 5 μL
2 -nanostructures -ligand 1 μL 1 μM attachment DNA 2 μL 2 μL 1 μL 500 U/mL 14 μL
3 -nanostructures -attachment 1 μL 1 μM oligo-ligand 2 μL 2 μL 1 μL 500 U/mL 14 μL
4 -nanostructures -enzyme 1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand 2 μL 2 μL 0 μL 14 μL
5 -nanostructures 1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand 2 μL 2 μL 1 μL 500 U/mL 13 μL
6 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
7 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
8 -oligos 2.25 μL 44 nM p7308 2 μL 2 μL 1 μL 500 U/mL 13.75 μL
9 -enzyme 10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.C 2 μL 2 μL 0 μL 6 μL
10 -ligand 10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.A 2 μL 2 μL 1 μL 500 U/mL 5 μL

results/discussion

  • lane 2: attachment DNA shows two bands (at 20 bp and 30 bp)
    • unusual, but not fatal
  • lane 3: oligo-ligand shows at 30 bp
  • lane 4: ds construct shows at 85 bp, excess oligo-ligand at 30 bp
  • lane 5: digested construct shows at 15 bp, undigested traces at 85 bp, excess oligo ligand at 30 bp
    • digest on ds construct (postive control) is successful
  • lanes 6 and 9: no visible difference between nanostructures when enzyme is added (lane 6) and when it is not added (lane 9)
    • implies that oligo streaks are unremoved oligos
  • lanes 6 and 10: no visible difference between nanostructures when oligo-ligand was folded in (lane 6) and when it was not (lane 10)
    • again, implies that oligo streaks are unremoved oligos
  • lane 7: most oligos appear to be removed
  • lane 8: scaffold is not digested
    • thank goodness

conclusions

  • perhaps need stronger digest conditions (more enzyme?) and more nanostructures
  • purer samples are needed, but this is not impossible (lane 7 looks great)
    • repeat PEG precipitation on resuspended samples?

Protection assay again

  • change: doubled enzyme amount, doubled amount of product loaded into gel
  • change: used 7% PEG-ppt'd 5.0.A and 5.0.C, maybe they'll be purer
  • used nanostructures folded above that were purified with 7% (A, C) or 8% (D) PEG, 0.5 M NaCl
  • mixed the following reactions, adding enzyme last
  • incubated at 37[[:Category:{{{1}}}|{{{1}}}]] for 60 min.
  • 20 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

[[|thumb|18% Tris-gly PAGE]]

lane trial DNA 10x NEBuffer 4 10x BSA AscI water
1 10 bp+ ladder 5 μL
2 -nanostructures -ligand 1 μL 1 μM attachment DNA 2 μL 2 μL 2 μL 500 U/mL 13 μL
3 -nanostructures -attachment 1 μL 1 μM oligo-ligand 2 μL 2 μL 2 μL 500 U/mL 13 μL
4 -nanostructures -enzyme 1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand 2 μL 2 μL 0 μL 14 μL
5 -nanostructures 1 μL 1 μM attachment DNA, 1 μL 1 μM oligo-ligand 2 μL 2 μL 2 μL 500 U/mL 12 μL
6 outward-facing ligands 10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.C 2 μL 2 μL 2 μL 500 U/mL 4 μL
7 inward-facing ligands 10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.D 2 μL 2 μL 2 μL 500 U/mL 4 μL
8 -oligos 2.25 μL 44 nM p7308 2 μL 2 μL 2 μL 500 U/mL 12.75 μL
9 -enzyme 10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.C 2 μL 2 μL 0 μL 6 μL
10 -ligand 10 μL 10 nM 8%-PEG-purified, ligand-incubated c5.0.A 2 μL 2 μL 2 μL 500 U/mL 4 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.
  • Gel:


Lane Component Amount
1 1kb ladder 10uL
2 p7308 9uL
3 6hb wash-supernatant 30uL
4 c5.0.8b wash-supernatant 30uL
5 c5.0.Eb wash-supernatant 30uL
6 c5.0.Fb wash-supernatant 30uL
7 6hb final-supernatant (?) 30uL
8 c5.0.8b final-supernatant (?) 30uL
9 c5.0.Eb final-supernatant (?) 30uL
10 c5.0.Fb final-supernatant (?) 30uL


  • Results:
    • As would concur with the suspicion that the wrong tubes had been Speedvaced, none of the four lanes of final-supernatant show any elute at all. The fact that there is no band in the biotinylated oligo final-supernatant lane (ie. lane 8) AND none in its wash-supernatant lane (ie. lane 4) suggests that the the biotinylated oligos were all bound by the streptavidin and then eluted off - but not into the "final-supernatant" tube. Thus, the experiment needs to be reconducted.


    • The bright bands that seem to be trapped in the wells of lanes 3 and 5 (ie. 6hb wash and Eb wash) suggest that some damage was suffered by the nanoboxes during the Speedvacing. There are two fainter bands in each of these lanes, one running slightly higher (slower) than the p7308, one running lower (faster) than the p7308. This could be undamaged nanostructure - however, since no c5.0.A (c5.0 barrel) was run as a control, no definite conclusions can be drawn.
      • The fact that lane 5 (Eb wash) shows such a large amount of nanoboxes, whereas lane 6 (Fb wash) does not, implies that Eb ("outside" biotinylation) was not bound by the streptavidin while Fb ("inside" biotinylation) was, as was true in the last gel (previously thought to be due to a mix up of the tubes). The most likely conclusion to be drawn is that there was mix up in the creation of the working stocks or pre-working stocks: Eb must truly be biotinylated on the inside, Fb on the outside, which may be due to a mistake in mixing c5.0.8b instead of c5.0.9b into Fb and vice versa, or because the oligos intended for the c5.0.8b pre-working stock were confused with those for c5.0.9b and vice versa.
        • Upon further investigation:
          • The sequences for c5.0.8.1 and c5.0.9.1 on the tube match those on the final oligo list for c5.0.
          • Oligo 7 (pre-split c5.0.8.1) correctly has an OUT aptamer site, and oligo 33 (pre-split c5.0.9.1) correctly has an IN aptamer site.
          • The best conclusion is that the pre-working or working stocks were incorrectly mixed.
        • Either way, correct working stocks and correct pre-working stocks must be made up and the former ones thrown out.


    • There are no bands in lane 6 (Fb) other than one that runs at the same speed as p7308, and this considerably fainter than the total brightness in lane 5 (Eb), suggesting that the nanoboxes have been bound by streptavidin.

This is a positive indicator that the protection assay worked, as are the other results from this gel, but to be certain, the experiment must be run again with the final-supernatants present.

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