June 12 (Monday)

Folding DNA Nanostructures

1. Working Stocks

   44 nM scaffold (20 microL)
   0.99 microM of each oligo

2. Protocol

   - goal: 10 nM scaffold w/ 100 nM each oligo, total rxn vol of 20 microL
   - calculations:
      scaffold = (10 nM)/(44 nM) * 20 microL = 4.5 microL
      oligos = (100 nM)/(990 nM) * 20 microL = 2 microL
   - reaction mixture:
      4.5 microL p7308 scaffold
      2 microL oligos
      2 microL 10x folding buffer (500 mM HEPES ph 7.5, 500 mM NaCl, 100 mM MgCl2)
      11.5 microL dH2O
   - annealing times:
      90 dC, 5'
      65 dC, 20'
      55 dC, 20'
      45 dC, 20'
      37 dC, 30'
   - neg. controls: 1 replacing oligos w/ water, 1 replacing scaffold w/ water

Transformation

1. Plasmids

   R0010: lac operon promoter
   E7104: T7 promoter + GFP
   E0241: GFP

2. Protocol

   - let chemically competent OneShot Top10 cells thaw on ice
   - added appropriate DNA to cells and tapped gently to mix
   - let sit on ice for 20 min
   - heat shocked @ 42 dC for 30"
   - let cool on ice for 2 min
   - added 200 microL SOC media
   - shook @ 37 dC for 1 hr
   - pipetted and spread onto agar plates treated w/ ?
   - incubated @ 37 dC overnight agar side-up