IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-11

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Thoughts/ramblings/goals/questions/general frustrations

The results of yesterday's experiment show that Microcon filtration gives low yields and the PEG precipitation (at least at 10%) damages nanostructures regardless of folding conditions.

Questions

  • Are Microcon yields unacceptably low, or are they acceptable? (Can we use the NanoDrop to quantify our yield?)
    • Gels are much better for quantifying yield - you can try both and see how they compare.
  • Low concentrations of PEG should precipitate large nanostructures. Will some smaller concentration of PEG not harm nanostructures formed under some folding conditions?
    • Our August 2 experiment showed that even low concentrations of PEG damage nanostructures folded under "standard" conditions (10x oligos, 10 mM MgCl2).


Gigundo PEG precipitation

  • goal: test 0% to 8% PEG precipitations with nanostructures folded under all six folding conditions from yesterday
  • optimistic hypothesis: nanostructures folded with higher concentrations of oligos and/or MgCl2 will show less damage after treatment with PEG

Protocol: prepare the following 30 samples.

Gel 1
Gel 1
Gel 2
Gel 2
Trial Final PEG % Gel Lanes 20% PEG (μL) 5 M NaCl (μL) Nanostructures (μL) water (μL) Total volume (μL)
1-00%110552050
1-22%135551550
1-44%1510551050
1-66%171555550
1 kb+ ladder1910
p730811010
1-88%1112055050
2-00%1130552050
2-22%1155551550
2-44%11710551050
2-66%1191555550
2-88%1212055050
3-00%1230552050
3-22%1255551550
3-44%12710551050
1 kb+ ladder12910
p730813010
3-66%1311555550
3-88%1332055050
4-00%1350552050
4-22%1375551550
4-44%13910551050
4-66%211555550
4-88%232055050
5-00%250552050
5-22%275551550
1 kb+ ladder12910
p730813010
5-44%21110551050
5-66%2131555550
5-88%2152055050
6-00%2170552050
6-22%2195551550
6-44%22110551050
6-66%2231555550
6-88%2252055050
1 kb+ ladder12910
p730813010
  • incubate on ice for 15 min.
  • spin at 16 k rcf at 4[[:Category:{{{1}}}|{{{1}}}]] for 10 min.
  • carefully pipet off supernatant
  • resuspend "pellet" in 20 μL of respective folding buffer
  • load resuspended pellets in odd-numbered lanes of 2% TBE agarose gel supplemented to 10 mM MgCl2
  • load 30 μL (of 50 μL) of supernatant into adjacent even-numbered lanes (e.g., trial 1-0 has pellet in lane 1 and supernatant in lane 2)
  • run at 60V for 1 h

Results/discussion

  • PEG precipitations appeared to have failed: no oligos were separated
  • curiously, the dye in the "supernatant" lanes ran at about 2/3 of the speed of the dye in the "pellet" lanes, and it gave a smear and not a band

Incubation of 3.2.E with thrombin beads

  • Goal: test if we can detect the binding of a nanostructure with outside aptamers to thrombin beads.

100 uL 10 nM 3.2.E or 100 uL 10 nM mix of 6.4. H/I was incubated with 250 uL thrombin beads (supplied as a 50% slurry). 3.2.E has outside aptamer sequences while 6.4.H/I do not. Following a 30 minute incubation, the beads were washed. The beads were then eluted by incubating with 250 uL 50% w/v free thrombin for 30 minutes. Washes and elutions were run on a 2% agarose gel for 60 minutes at 60V.

Lane Contents
01kb+ DNA ladder
26.4. H/I wash
46.4. H/I elution
63.2.E wash
83.2.E elution

Image:nano811beadgel.jpg

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