IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-18

Questions

Design 2

• How big does a 2-ply lid need to be to span a 30-helix barrel?
• Can we use two scaffolds in the design?
  • 2x42bp repeats in barrel
  • 30-helix bundle with 30*(2*42+12) repeats = 2880 bases
  • two-ply-lid: 36 * (2*42+12) = 3456 * 2 = 6912
  • using single scaffold for 2880-base 30hb: 9792 bases total = $783.36
  • 3x42bp repeats in barrel
  • 30-helix bundle with 30*(3*42+12) repeats = 4140 bases
  • two-ply-lid: 36 * (2*42+12) = 3456 * 2 = 6912
  • using single scaffold for 4140-base 30hb: 11052 bases total = $884.16

• Will unused scaffold interfere with proper folding of lid & barrels
  • Should we use two M13-based scaffolds, or try to make ΦX174 to have less slack?
  • Can we easily get ΦX174? fermentas

Protection assay

Questions

• Can we digest streptavidin with Qiagen proteinase K and/or Qiagen protease?
• What alternative proteases can we use?
  • We're going to try trypsin
• What is our initial protocol?

Proteinase K protocol

• Wu's digestion protocol [1]: Proteinase K Digestion of Streptavidin and Its Muteins—Purified streptavidin and its muteins (30 μM monomer) were treated with proteinase K (Invitrogen, 5 μM) for 15 min at 30[[:Category:{{{1}}}|{{{1}}}]] in 50 mM Tris-HCl containing 5 mM CaCl2, pH 8.0. The reaction was stopped by precipitation with trichloroacetic acid (18). Boiled samples of precipitated proteins were resolved by reducing SDS-PAGE. The same analysis was performed with streptavidin samples treated with biotin (1 mM final concentration) prior to proteinase K digestion.
• A possible protocol:
  • Streptavidin tetramer (20 μM) is incubated with 3'-biotinylated oligo (10 μM) for 5 min. at room temperature (28[[:Category:{{{1}}}|{{{1}}}]] today, whew)
  • Streptavidin tetramer (one expt. biotin-treated, one expt. biotin-untreated), is treated with 5 μM proteinase K for 15 min at 30[[:Category:{{{1}}}|{{{1}}}]] in 50 mM Tris-HCl containing 5 mM CaCl2, pH 8.0
  • Reaction is stopped by EtOH precipitation of DNA, or by filtration through a Qiagen column