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

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

PEG precipitation

• Goal: repeat titration of PEG precipitation conditions for folded containers
• 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)

• 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