User:Vlau: Difference between revisions
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==='''06.13.06: Tues.'''=== | ==='''06.13.06: Tues.'''=== | ||
====Gel Electrophoresis w/ 2% Gel==== | |||
1. Protocol | |||
- mixed 2 g Ultrapure agarose w/ 100 mL 1X TBE in plastic flask | |||
- heated for 1.5 min in microwave w/ top loosely screwed on | |||
- added 3 microL EtBr | |||
- poured into gel frame and let set for 20 min | |||
- placed into running box and submerged w/ TBE | |||
- wells loaded | |||
- ran gel @ 130 V, 45 min | |||
2. Samples | |||
L. 1: 1 kB ladder | |||
L. 2 and 3: DNA nanostructure | |||
L. 4 and 5: neg control w/ no oligonucleotides | |||
L. 6 and 7: neg control w/ no scaffold | |||
L. 8: 1 kB ladder | |||
loading dye: BTB dye 50% glycerol + 10X TBE | |||
ladder: 10 microL + 1 microL dye | |||
samples: 10 microL + 1 microL dye | |||
3. Image | |||
[[Image:Pic 1.jpg]] | |||
====Transformation P. 2==== | |||
1. Protocol | |||
- prepared 2 liquid cultures for each transformation | |||
- added 50 microL of 5 mg/mL amp to 5 mL LB per culture | |||
- shook @ 180 rpm, 37 dC overnight | |||
Revision as of 21:58, 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
06.13.06: Tues.
Gel Electrophoresis w/ 2% Gel
1. Protocol
- mixed 2 g Ultrapure agarose w/ 100 mL 1X TBE in plastic flask - heated for 1.5 min in microwave w/ top loosely screwed on - added 3 microL EtBr - poured into gel frame and let set for 20 min - placed into running box and submerged w/ TBE - wells loaded - ran gel @ 130 V, 45 min
2. Samples
L. 1: 1 kB ladder L. 2 and 3: DNA nanostructure L. 4 and 5: neg control w/ no oligonucleotides L. 6 and 7: neg control w/ no scaffold L. 8: 1 kB ladder loading dye: BTB dye 50% glycerol + 10X TBE ladder: 10 microL + 1 microL dye samples: 10 microL + 1 microL dye
3. Image
Transformation P. 2
1. Protocol
- prepared 2 liquid cultures for each transformation - added 50 microL of 5 mg/mL amp to 5 mL LB per culture - shook @ 180 rpm, 37 dC overnight
