IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-1

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To do today

  • ordering
    • new oligo ligands
    • AscIII (if not already ordered
  • PEG precipitations
    • repeat experiment, run on PA gel, run on agarose gel
  • SYBR gold
    • read technical specs
    • determine lower threshold of imaging

Ordering

Order form for v5.0 latches, v3.2 attachment oligos, and v5.0 attachment oligos

  • v5.0 latches and v3.2 and v5.0 ligand-attachment oligos ordered
  • forgot to order the ligands - will do tomorrow, along with the clarified v4.0 ligand-attachment oligos

SYBR gold testing

Trial 1

  • goal: to determine the minimum amount of DNA that can be visualized with SYBR Gold
  • methods: 12% PA gel with lanes listed below, run for 30 min. at 130 V, then stained with Invitrogen SYBR Gold.
    • prepared 1x solution: 10 μL thawed 10,000x SYBR gold in 100 mL TBE, stored in light-proof container at 4[[:Category:{{{1}}}|{{{1}}}]].
lane DNA (3.2.7.2b)
(12.5 pg DNA / fmol)
(μL) (fmols) (pg)
1 15 μL 1 μM 15,000 187,500
2 10 μL 1 μM 10,000 125,000
3 5 μL 1 μM 5,000 62,500
4 2.5 μL 1.0 μM 2,500 31,250
5 1 μL 1.0 μM 1,000 12,500
6 5 μL 0.1 μM 500 6,250
7 2.5 μL 0.1 μM 250 3,125
8 1 μL 0.1 μM 100 1,250
9 5 μL 0.01 μM 50 625
10 2.5 μL 0.01 μM 25 313
11 1 μL 0.01 μM 10 125
12 10 μL 1kb+ ladder
  • stained with 1x SYBR Gold for 50 min.
  • results
    • ladder stained brilliantly, visible both under SYBR Gold filter (and less so under EtBr filter)
    • no other DNAs showed, including after EtBr soak
    • dye was only halfway down gel, so ss oligos probably didn't run off
    • will try again with non biotinylated oligo

Trial 2

  • goal: try again, different DNA
  • run on August 2

Container 5.0 lid refolding

  • Yesterday's gel result indicated that folding two lids from a single scaffold didn't work well
  • Goal: fold c5.0 lids using separate scaffolds
2% agarose gel, 0.5 mg/mL EtBr
0.5x TBE, 11 mM MgCl2
Lane Contents Loading Buffer
0 1kb DNA ladder (4 μL)
1 naked p7704 (10 μL) AGLB (2 μL)
2 c5.0 lid 1 (10 μL) AGLB (2 μL)
3 c5.0 lid 2 (10 μL) AGLB (2 μL)
4 c5.0 lid 1+2 (10 μL) AGLB (2 μL)
  • Lid 1 seems to be causing the smearing
  • Lid 1+2 looks a lot better in this trial for some reason
  • Proper p7572 scaffold may improve things further

PEG precipitation

  • Goal: repeat titration of PEG precipitation conditions for folded containers
    • Katie and Val did one trial, Tiff and Matthew did another
  • Folding reactions
    • 2 samples of 6hb (100 μL final volume)
    • 4 samples of design 5 barrels (100 μL volume)
    • Mix the following
      • 10 uL 500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM MgCl2
      • 40 uL working stock 250 nM each oligo (100 nM each oligo final concentration)
      • 50 uL p7308 20 nM (10 nM final concentration)
    • Anneal from 80[[:Category:{{{1}}}|{{{1}}}]] to 20[[:Category:{{{1}}}|{{{1}}}]], -1[[:Category:{{{1}}}|{{{1}}}]] per min
  • PEG precipitations
  • Plan to use 200 μL final volume
  • Trying 4%, 6%, 8%, 10%, 12%, 14%
  • Add 40 μL 10 nM scaffold/100 nM oligo folded mix
  • Add 20% PEG solution (40 μL, 60 μL, 80 μL, 100 μL, 120 μL, 140 μL)
  • Add 5 M NaCl stock solution (20 μL)
  • Add as much water to each as it takes to get them to a 200 μL final volume
  • Incubate on ice for 15 minutes
  • Spin 16k rcf for 10 minutes
    • Tiff and Matt's trial: 0.2 mL PCR tubes broke through the 1.5 mL microcentrifuge tubes that had been holding them. Will try again using 1.5 mL PCR tubes for reaction mixtures.'
  • Pipette out supernatant into separate tube
  • Resuspend pellet in 1x folding buffer volume equal to the supernatant
  • Run equivalent volumes of pellet and sup on 2% agarose gel (+ 11 mM MgCl2)
  • Gel Analysis
    • Analyze on 2% Mg agarose gel (0.5x TBE, 11 mM MgCl2, 0.5 μg/mL EtBr)
    • Load 1kb ladder (1 lane)
    • Load c5 supernatant, pellet for 0%, 4%, 6%, 8%, 10%, 12%, 14% (13 lanes)
    • Load 6hb supernatant, pellet for 0%, 4%, 14% (5 lanes)
    • 19 lanes total

Katie and Val's results:

2% agarose gel, 0.5 mg/mL EtBr
0.5x TBE, 11 mM MgCl2
Lane Contents Loading Buffer
0 1kb DNA ladder (4 μL)
1 6hb untreated (10 μL) AGLB (2 μL)
2 6hb 4% PEG supernatant (10 μL) AGLB (2 μL)
3 6hb 4% PEG pellet (10 μL) AGLB (2 μL)
4 6hb 14% PEG supernatant (10 μL) AGLB (2 μL)
5 6hb 14% PEG pellet (10 μL) AGLB (2 μL)
6 c5 barrel untreated (10 μL) AGLB (2 μL)
7 c5 barrel 4% PEG supernatant (10 μL) AGLB (2 μL)
8 c5 barrel 4% PEG pellet (10 μL) AGLB (2 μL)
9 c5 barrel 6% PEG supernatant (10 μL) AGLB (2 μL)
10 c5 barrel 6% PEG pellet (10 μL) AGLB (2 μL)
11 c5 barrel 8% PEG supernatant (10 μL) AGLB (2 μL)
12 c5 barrel 8% PEG pellet (10 μL) AGLB (2 μL)
13 c5 barrel 10% PEG supernatant (10 μL) AGLB (2 μL)
14 c5 barrel 10% PEG pellet (10 μL) AGLB (2 μL)
15 c5 barrel 12% PEG supernatant (10 μL) AGLB (2 μL)
16 c5 barrel 12% PEG pellet (10 μL) AGLB (2 μL)
17 c5 barrel 14% PEG supernatant (10 μL) AGLB (2 μL)
18 c5 barrel 14% PEG pellet (10 μL) AGLB (2 μL)
File:IGEM06-SD-0600801c.jpg
2% agarose gel, 0.5 mg/mL EtBr
0.5x TBE, 11 mM MgCl2
Lane Contents Loading Buffer
0 1kb DNA ladder (4 μL)
1 c5 barrel untreated (10 μL) AGLB (2 μL)
2 c5 barrel 4% PEG supernatant (10 μL) AGLB (2 μL)
3 c5 barrel 4% PEG pellet (10 μL) AGLB (2 μL)
4 c5 barrel 5% PEG supernatant (10 μL) AGLB (2 μL)
5 c5 barrel 5% PEG pellet (10 μL) AGLB (2 μL)
6 c5 barrel 6% PEG supernatant (10 μL) AGLB (2 μL)
7 c5 barrel 6% PEG pellet (10 μL) AGLB (2 μL)
8 c5 barrel 10% PEG supernatant (10 μL) AGLB (2 μL)
9 c5 barrel 10% PEG pellet (10 μL) AGLB (2 μL)
10 c5 barrel 14% PEG supernatant (10 μL) AGLB (2 μL)
11 c5 barrel 14% PEG pellet (10 μL) AGLB (2 μL)


EM imaging

High Concentrations of Nanoboxes, Day 2

  • Qiagen gel purified 3rxns of each Eb and Gb. Used 2% agarose Mg2+-supplemented gel. Eluted each column (used 2/3rxns, so as to try to avoid overloading the column filter) into 40uL of EB, leaving 3rxns worth of nanoboxes (approx 200*3fmol, or 600fmol of biotin binding sites) in 80uL of solution.
    • scaffold (2nd lane) and folded Eb and Gb (3rd-14th lane) run differently - indicates folding worked
  • PEG precipitated at 12% total concentrations. Accidentally tossed out the supernatant. Reconstituted pellet in 80ul of 1x folding buffer, to try to get comparable concentrations of nanoboxes.
  • Ran 10ul of each reaction on 2% agarose Mg2+-supplemented gel (wanted to run with scaffold, but none left in the lab). Saw bright bands for PEG-precipitated boxes, none for gel-purified. Mg2+ in the original gel might have interfered with column interaction.
    • gel purification shows nothing - didn't work, or concentrations too low
      • NB: Shawn has said that the Qiagen gel purification reagents will cause unfolding of the boxes - gel purification should therefore no longer be used for nanoboxes.
    • PEG-pelleted nanoboxes look bright (even though this is only 10uL of a 3-rxn condensation - in essence, 3/8 of a normal 40uL-rxn)