User:Vlau: Difference between revisions
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===Lab Notebook=== | ===Lab Notebook=== | ||
==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 | |||
[[June 12 (Monday)]] | [[June 12 (Monday)]] |
Revision as of 21:52, 15 June 2006
Valerie Lau
vlau@fas.harvard.edu/vhtlau@gmail.com
Lab Notebook
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