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
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