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

From OpenWetWare
Jump to: navigation, search

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 2
Trial Final PEG % Gel Lanes 20% PEG (μL) 5 M NaCl (μL) Nanostructures (μL) water (μL) Total volume (μL)
1-0 0% 1 1 0 5 5 20 50
1-2 2% 1 3 5 5 5 15 50
1-4 4% 1 5 10 5 5 10 50
1-6 6% 1 7 15 5 5 5 50
1 kb+ ladder 1 9 10
p7308 1 10 10
1-8 8% 1 11 20 5 5 0 50
2-0 0% 1 13 0 5 5 20 50
2-2 2% 1 15 5 5 5 15 50
2-4 4% 1 17 10 5 5 10 50
2-6 6% 1 19 15 5 5 5 50
2-8 8% 1 21 20 5 5 0 50
3-0 0% 1 23 0 5 5 20 50
3-2 2% 1 25 5 5 5 15 50
3-4 4% 1 27 10 5 5 10 50
1 kb+ ladder 1 29 10
p7308 1 30 10
3-6 6% 1 31 15 5 5 5 50
3-8 8% 1 33 20 5 5 0 50
4-0 0% 1 35 0 5 5 20 50
4-2 2% 1 37 5 5 5 15 50
4-4 4% 1 39 10 5 5 10 50
4-6 6% 2 1 15 5 5 5 50
4-8 8% 2 3 20 5 5 0 50
5-0 0% 2 5 0 5 5 20 50
5-2 2% 2 7 5 5 5 15 50
1 kb+ ladder 1 29 10
p7308 1 30 10
5-4 4% 2 11 10 5 5 10 50
5-6 6% 2 13 15 5 5 5 50
5-8 8% 2 15 20 5 5 0 50
6-0 0% 2 17 0 5 5 20 50
6-2 2% 2 19 5 5 5 15 50
6-4 4% 2 21 10 5 5 10 50
6-6 6% 2 23 15 5 5 5 50
6-8 8% 2 25 20 5 5 0 50
1 kb+ ladder 1 29 10
p7308 1 30 10
  • 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
0 1kb+ DNA ladder
2 6.4. H/I wash
4 6.4. H/I elution
6 3.2.E wash
8 3.2.E elution

Nano811beadgel.jpg