IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-25

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PEG fractionation

  • Goal: to get cleaner purification of oligos away from nanostructures and to increase the volume of purified nanostructures we have for protection assays. Given the results of 8-23 I'm working with a new batch of folded design 5 and trying to see if the double bands and trouble moving through the gel were simply artifacts of that particular folding or if they are larger problems that need to be addressed.
  • Nanostructures:
    • using 30mM MgCl2, 1x oligos (seems to work best given previous results).
    • designs: c5.0.A, c5.0.C, c5.0.D
  • PEG: 8%, 10%. Total volume in each is 100 μL
    • 8 % Cocktail:
     40 μL 20% PEG
     10 μL 5M NaCl
     10 μL water
     40 μL Nanostructures (add last)
    • 10 % Cocktail:
     50 μL 20% PEG
     10 μL 5M NaCl
     40 μL Nanostructures (add last)
  • incubate on ice for 15 min.
  • spin at 16 k rcf at 4[[:Category:{{{1}}}|{{{1}}}]] for 10 min.
  • carefully pipette off supernatant
  • resuspended "pellet" in 1x folding buffer. for now, resuspend in original total volume (100 μL but may resuspend in less in the future to improve protection assay results)
    • note: add PEG first, nanostructures last; mix using tapping after everything added. let sit for ~5 min. before putting it on ice
Lane contents
gel1-1 ladder
gel1-2 scaffold
gel1-3 5.0.A unpurified
gel1-4 5.0.A 8% pellet 1
gel1-5 5.0.A 8% supernatant 1
gel1-6 5.0.A 8% pellet 2
gel1-7 5.0.A 8% supernatant 2
gel1-8 5.0.A 10% pellet 1
gel1-9 5.0.A 10% supernatant 1
gel1-10 5.0.A 10% pellet 2
gel1-11 5.0.A 10% supernatant 2
gel1-12 5.0.C unpurified
gel1-13 5.0.C 8% pellet 1
gel1-14 5.0.C 8% supernatant 1
gel1-15 5.0.C 8% pellet 2
gel1-16 5.0.C 8% supernatant 2
gel1-17 5.0.C 10% pellet 1
gel1-18 5.0.C 10% supernatant 1
gel1-19 5.0.C 10% pellet 2
gel1-20 5.0.C 10% supernatant 2
gel2-1 ladder
gel2-2 5.0.D unpurified
gel2-3 5.0.D 8% pellet 1
gel2-4 5.0.D 8% supernatant 1
gel2-5 5.0.D 8% pellet 2
gel2-6 5.0.D 8% supernatant 2
gel2-7 5.0.D 10% pellet 1
gel2-8 5.0.D 10% supernatant 1
gel2-9 5.0.D 10% pellet 2
gel2-10 5.0.D 10% supernatant 2


Streptavidin Bead Depletion: Investigation of Fb

Strep Treatment Protocol

  • Same as that done Wednesday, only with just the c5.0.A and c5.0.Fb folded yesterday, and the c5.0.Fb folded by Katie on 8.22.
  • Other modification: everything was rediluted up to 35uL instead of 40uL in 30mM 1x folding buffer.

Gel

  • NB: Accidentally ran old-folded Fb before new-folded Fb in the untreated trial. New-folded Fb was loaded into the gel before old-folded Fb in every other trial.
  • 2% agarose, 0.5xTBE supplemented to 10mM MgCl2; 5uL EtBr
  • run @ 100V for 1hr in 0.5xTBE supplemented to 10mM MgCl2
  • 2uL 10x TBE/glycerol loading dye for each well



Lane Component Test Condition Amount
1 1kb+ ladder - 10uL
2 p7308 (~42nM) - 9uL
3 c5.0.A barrel (lidless) Untreated 10uL
4 c5.0.Fb inside biotinylated barrel (folded 8.22) Untreated 10uL
5 c5.0.Fb inside biotinylated barrel (folded 8.24) Untreated 10uL
6 barrel (lidless) Beaded ~38uL
7 inside biotinylated barrel (folded 8.24) Beaded ~38uL
8 inside biotinylated barrel (folded 8.22) Beaded ~38uL
9 barrel (lidless) Free-strep ~38uL
10 inside biotinylated barrel (folded 8.24) Free-strep ~38uL
11 inside biotinylated barrel (folded 8.22) Free-strep ~38uL
12 barrel (lidless) Free, bead ~38uL
13 inside biotinylated barrel (folded 8.24) Free, bead ~38uL
14 inside biotinylated barrel (folded 8.22) Free, bead ~38uL


Results

  • Though it seems that the 8.24 Fb may be better folded than the 8.22 Fb, as seen by the decreased amount of streaking and material trapped in the well in lane 5 as opposed to lane 4, the 8.24 Fb is still taken up by the streptavidin beads (no band in lane 8). This indicates either:
    • 1. There is some misfolding property of Fb that exposes the inside environment of the barrel to the outside environment of the beaded environment.
      • This would suggest steps to ascertain reasons for this misfolding:
        • Looking at the pre-working stocks that differ from Eb
        • Analyzing the sequences of the oligos involved to make sure that there were no mistakes made
        • Remixing the pre-working stocks involved, in case the wrong wells in the plates were used in making these originally
        • Remixing Fb
    • 2. Streptavidin, though bound to beads, can still reach far enough into the barrel to bind (and thus remove from the supernatant) inside biotin.
        • This is possible. As detailed earlier, the dimensions of the streptavidin molecule (in conjunction with the lack of detail about where on the streptavidin molecule is linked to the bead in relation to the biotin-binding site) could technically allow it to reach the biotins, which are hanging off the side of the barrel by 3 Ts, or (3*0.34nm =) roughly 1nm. This is in addition to the actual length of the biotin molecule, if the binding can be said to start at one end of the molecule.
        • Some of the inside biotinylated sites are only 3 7bp-sub-tokens away from the edge of the barrel, or (21*0.34nm=) 7.14nm.
        • If the 4-carbon linker, roughly 0.4nm in length, and this above information is taken into consideration, the streptavidin molecule could reach into the barrel roughly 4.8nm+0.4nm conservatively, 5.8+0.4nm at most. And the 3T-linker of 1nm and the length of the biotin molecule itself could conceivably give enough leeway for the streptavidin to bind internal sites.
      • If this is the case, we can order biotinylated oligos which exit the barrel closer to the middle of the barrel.
        • But before we take this step, we should try to ascertain whether this is actually the problem, as opposed to possibilities (1) or (3).
      • (Or we could wait till we get lids actually on the c5.0 barrel and try again - but this would assume that we'll be able to get the lids folded, attached correctly, and done before November. It seems like a bad idea to wait.)
    • 3. The biotins, though we designed them point out to the inside of the barrel (and we're certain that the aptamer sites were technically on the inside, as this is a double-ply barrel), they might be pushing out through the double-ply width of the barrel through holes between helices.
      • Because this is a double-ply barrel, this seems reasonably unlikely, but it is a possibility.