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

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Contents

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)
115 μL 1 μM15,000187,500
210 μL 1 μM10,000125,000
35 μL 1 μM5,00062,500
42.5 μL 1.0 μM2,50031,250
51 μL 1.0 μM1,00012,500
65 μL 0.1 μM5006,250
72.5 μL 0.1 μM2503,125
81 μL 0.1 μM1001,250
95 μL 0.01 μM50625
102.5 μL 0.01 μM25313
111 μL 0.01 μM10125
1210 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 EtBr0.5x TBE, 11 mM MgCl2
2% agarose gel, 0.5 mg/mL EtBr
0.5x TBE, 11 mM MgCl2
Lane Contents Loading Buffer
01kb DNA ladder (4 μL)
1naked p7704 (10 μL)AGLB (2 μL)
2c5.0 lid 1 (10 μL)AGLB (2 μL)
3c5.0 lid 2 (10 μL)AGLB (2 μL)
4c5.0 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 EtBr0.5x TBE, 11 mM MgCl2
2% agarose gel, 0.5 mg/mL EtBr
0.5x TBE, 11 mM MgCl2
Lane Contents Loading Buffer
01kb DNA ladder (4 μL)
16hb untreated (10 μL)AGLB (2 μL)
26hb 4% PEG supernatant (10 μL)AGLB (2 μL)
36hb 4% PEG pellet (10 μL)AGLB (2 μL)
46hb 14% PEG supernatant (10 μL)AGLB (2 μL)
56hb 14% PEG pellet (10 μL)AGLB (2 μL)
6c5 barrel untreated (10 μL)AGLB (2 μL)
7c5 barrel 4% PEG supernatant (10 μL)AGLB (2 μL)
8c5 barrel 4% PEG pellet (10 μL)AGLB (2 μL)
9c5 barrel 6% PEG supernatant (10 μL)AGLB (2 μL)
10c5 barrel 6% PEG pellet (10 μL)AGLB (2 μL)
11c5 barrel 8% PEG supernatant (10 μL)AGLB (2 μL)
12c5 barrel 8% PEG pellet (10 μL)AGLB (2 μL)
13c5 barrel 10% PEG supernatant (10 μL)AGLB (2 μL)
14c5 barrel 10% PEG pellet (10 μL)AGLB (2 μL)
15c5 barrel 12% PEG supernatant (10 μL)AGLB (2 μL)
16c5 barrel 12% PEG pellet (10 μL)AGLB (2 μL)
17c5 barrel 14% PEG supernatant (10 μL)AGLB (2 μL)
18c5 barrel 14% PEG pellet (10 μL)AGLB (2 μL)
Image:IGEM06-SD-0600801c.jpg
2% agarose gel, 0.5 mg/mL EtBr
0.5x TBE, 11 mM MgCl2
Lane Contents Loading Buffer
01kb DNA ladder (4 μL)
1c5 barrel untreated (10 μL)AGLB (2 μL)
2c5 barrel 4% PEG supernatant (10 μL)AGLB (2 μL)
3c5 barrel 4% PEG pellet (10 μL)AGLB (2 μL)
4c5 barrel 5% PEG supernatant (10 μL)AGLB (2 μL)
5c5 barrel 5% PEG pellet (10 μL)AGLB (2 μL)
6c5 barrel 6% PEG supernatant (10 μL)AGLB (2 μL)
7c5 barrel 6% PEG pellet (10 μL)AGLB (2 μL)
8c5 barrel 10% PEG supernatant (10 μL)AGLB (2 μL)
9c5 barrel 10% PEG pellet (10 μL)AGLB (2 μL)
10c5 barrel 14% PEG supernatant (10 μL)AGLB (2 μL)
11c5 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)
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