IGEM:Harvard/2006/vlau/Notebook/2006-6-12
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Folding DNA Nanostructures
1. Working Stocks
44nM scaffold (20μL) 0.99μM of each oligo
2. Protocol
- goal: 10 nM scaffold w/ 100 nM each oligo, total rxn vol of 20μL - calculations: scaffold = (10nM)/(44nM) * 20μL = 4.5μL oligos = (100nM)/(990nM) * 20μL = 2μL - reaction mixture: 4.5μL p7308 scaffold 2μL oligos 2μL 10x folding buffer (500mM HEPES ph 7.5, 500mM NaCl, 100mM MgCl2) 11.5μL 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 200μL SOC media - shook @ 37dC for 1 hr - pipetted and spread onto agar plates treated w/ ? - incubated @ 37dC overnight agar side-up