IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-14

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Contents

Goals for today

Microcon Purification Tweaking

  • repeat Friday's mega PEG ppt on 5.0 (?)
  • Micron experiments with 0.1% and 0.01% SDS in buffer
    • ...and use 1x folding buffer and not water for washes
    • also: perform control expt with 10 bp+ ladder, since according to Millipore documentation, the filter should retain ds DNAs longer than 100 bp

Streptavidin-Bead "Protection" Assay on Inside- and Outside-Biotinylated c5.0

  • NB: no good purification of nanostructure from oligo has been achieved, but gel separation after elution should differentiate formerly bead-bound oligos from formerly bead-bound nanostructures

Redux of [Mg++], [oligos]



Mg2+, Oligo-Concentration Titration w/ c5.0

Goals

  • vary folding conditions ([MgCl2] and [oligo]) in order to determine best folding conditions for c5.0
  • determine most efficient purification protocol (Microcon vs. PEG) based on recovery yields

Protocol

1. Working Stock Concentration

  • concentrated 6 tubes of 96 μL c5.0D.L (no latches, outside-bound ligand) in Vacufuge so that [oligo]= 250nM * 6 = 1.5 μM

2. Folding Rxns

  • used three different folding buffers varying [MgCl2]
  • used two different [oligo concentrations]: 250 nM from the unconcentrated working stock, 1.5 μM from above
  • folding conditions: 80[[:Category:{{{1}}}|{{{1}}}]] for 2 min., decrease 1[[:Category:{{{1}}}|{{{1}}}]] every 2 min. for 59 more times

Notes

  • a,b - a and b of each are the same, just two different tubes.
  • Oligos - 250 nM is 1x oligos, 1.5 {{um} is 6 oligos
Trial Oligos p7308 (44 nM) Folding Buffer (10x) Water
1a,b16 μL 250 nM9 μL4 μL 100 mM MgCl211 μL
2a,b16 μL 1.5 μM9 μL4 μL 100 mM MgCl211 μL
3a,b16 μL 250 nM9 μL4 μL 200 mM MgCl211 μL
4a,b16 μL 1.5 μM9 μL4 μL 200 mM MgCl211 μL
5a,b16 μL 250 nM9 μL4 μL 300 mM MgCl211 μL
6a,b16 μL 1.5 μM9 μL4 μL 300 mM MgCl211 μL

Microcon w/ detergent

  • add 20 μL given nanostructure to center of YM-50 Micrcon tube
  • add 480 μL given folding buffer, microcentrifuge for 6 min. at 14k rcf, and repeat dilution and spinning 4 more times
  • yielded approx. 100 μL retentate, which was concentrated to 15 to 60 μL in a Vacufuge (about 30 min. at 45 [[:Category:{{{1}}}|{{{1}}}]]), depending on the sample
lane starting amt. of nanostructures wash buffer loaded onto gel
17 μL 1 kb+ ladder
22.25 μL p7308
310 μL unpurified 4.0.I
420 μL 6hb1x folding buffer (10 mM MgCl2)half of retentate
520 μL 6hb1x folding buffer (10 mM MgCl2) w/ 0.1% SDShalf of retentate
620 μL 4.0.I1x folding buffer (10 mM MgCl2)half of retentate
720 μL 4.0.I1x folding buffer (10 mM MgCl2) w/ 0.01% SDShalf of retentate
820 μL 4.0.I1x folding buffer (10 mM MgCl2) w/ 0.1% SDShalf of retentate
  • ran 2% agarose gel at 80 V for 1 h. Gel appears to be of such low qualitiy that the results are inconclusive (ladder isn't clear)
  • ran another 2% agarose gel at 60V for 1 h.
  • results/discussion
    • unclear why 6hb filtration failed (oligos were retained) (lane 4)
    • 0.1% SDS gives unusual/unknown smears (lanes 5 and 8)
      • under visible light, there are thin pink-red bands in the middle of these smears. is SDS breaking down the Microcon tube plastic?
    • 0.01% SDS possibly gives higher yields than no SDS (lane 7 vs. lane 6), but it is not conclusively better yield, and it is still very poor overall yield (lane 7 vs. lane 3)


Streptavidin Bead "Protection"

  • Goal:
    • Determine whether c5.0 functions like a container - in other words, can it "protect" biotinylated sites on the inside from being bound by streptavidin? In order to do this, we can use 1 micrometer-in-diameter magnetic streptavidin beads (ie. bigger in diameter than the hole of the c5.0 barrel, so the streptavidin bound to the bead won't be able to access the internal biotin).
    • If we remove the material that is not streptavidin-bound initially (hopefully, internally-biotinylated barrels) and then elute the material which was streptavidin-bound (externally-biotinylated barrels, free-floating biotinylated oligos), a gel run with the elutes will show DNA material for the externally-biotinylated structures.
  • Structural Justifications:
    • NEB's magnetic streptavidin beads are 1um in diameter, too large to fit into the 30nm-wide barrel of the c5.0.
    • However, conceivably you could imagine the streptavidin binding region being able to snake into the barrel, as streptavidin's dimensions are 54 x 58 x 48 angstroms, or 5.4 x 5.8 x 4.8 nm, which would allow it to fit in the barrel.
      • Biotinylated sites within the barrel are attached to oligos which are 3 Ts from the scaffold-oligo binding point, thus making this a possibility, but hopefully not a frequent one.
  • Issues:
    • No protocol found for the Fluka agarose streptavidin beads, and the NEB magnetic bead protocol's heating requirements seem unsuitable for keeping nanoboxes folded throughout the process
      • Nanoboxes must remain folded post-elution because they must be gel-distinguishable from eluted biotinylated oligos, which we have not been able to pre-purify out by other means.
    • Elution methods are of two types:
    • 1. Overload with competitors - either streptavidin or biotinylated oligos
      • But can't: biotin-streptavidin bond is so strong that dissociation by competition would theoretically take years
    • 2. Denature the streptavidin using a) formamide, b) phenol, c) SDS + boiling
      • But can't: all the conditions require heat that might damage the nanostructures (65[[:Category:{{{1}}}|{{{1}}}]] and up)
  • Solution:
    • Degrade streptavidin with trypsin
      • Trypsin must be in solution w/o EDTA, which would otherwise chelate all the Mg2+ in the DNA nanobox solution that is necessary to keeping it folded.
  • Protocol:
1. Incubate:
     5uL beads (binding capacity: 2pmol/1uL, thus, 10pmol - far greater than the available binding sites in the DNA nanobox solutions)
     35uL 1x folding buffer
     10uL test solution

           TEST SOLUTIONS:
           --------------
           a) H2O (ie. test = no biotin in solution)
           b) biotinylated oligos (c5.0.8(b)) - 250uM per oligo in pre-working stock = 1mM biotinylation - for 1.6pmol = 1.6uL - thus, 1.6uL + 8.4uL H2O must be added for test solution
           c) c5.0 E(b) (outside biotinylation) (was Microcon "purified" Tu 8.9) - ~2000fmol of binding sites/12.5uL, or 0.16pmol/ul - thus, 1.6pmol 
           d) c5.0 F(b) (inside biotinylation) (was Microcon "purified" Tu 8.9) - "

2. Mix
3. Pellet by drawing magnet down to bottom of tube (7 minutes using the old magnet)
4. Discard supernatant
5. Add 50uL 1x folding buffer

6. Repeat steps 2-5 three more times.

7. Trypsinize by adding to pellet:
      3uL trypsin (1mg/mL)
      27uL 1x folding buffer
8. Incubate 4hr-overnight @ 37 degrees C

9. Pellet by drawing magnet down to bottom of tube and remove supernatant to clean tube (1 minute, using the Magnetorack).
10. Run 20uL of each supernatant on 2% agarose gel (10mM MgCl2) for 1hr at 80V.
  • Gel:
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