IGEM:Harvard/2006/vlau/Notebook/2006-6-12: Difference between revisions
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37dC, 30' | 37dC, 30' | ||
- neg. controls: 1 replacing oligos w/ water, 1 replacing scaffold w/ water | - neg. controls: 1 replacing oligos w/ water, 1 replacing scaffold w/ water | ||
===''Transformation''=== | ===''Transformation''=== | ||
Latest revision as of 07:57, 2 August 2006
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
