IGEM:Harvard/2006/vlau/Notebook/2006-6-12

Folding DNA Nanostructures
1. Working Stocks 44nM scaffold (20) 0.99 of each oligo 2. Protocol - goal: 10 nM scaffold w/ 100 nM each oligo, total rxn vol of 20 - calculations: scaffold = (10nM)/(44nM) * 20 = 4.5 oligos = (100nM)/(990nM) * 20 = 2 - reaction mixture: 4.5 p7308 scaffold 2 oligos 2 10x folding buffer (500mM HEPES ph 7.5, 500mM NaCl, 100mM MgCl2) 11.5 dH2O - annealing times: 90dC, 5' 65dC, 20' 55dC, 20' 45dC, 20' 37dC, 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 @ 42dC for 30"   - let cool on ice for 2 min    - added 200 SOC media    - shook @ 37dC for 1 hr    - pipetted and spread onto agar plates treated w/ ?    - incubated @ 37dC overnight agar side-up