User:Vlau: Difference between revisions
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37 dC, 30' | 37 dC, 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==== | ||
1. Plasmids | 1. Plasmids | ||
Revision as of 21:57, 15 June 2006
Week 1
06.12.06: Mon.
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
