|
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| Line 13: |
Line 13: |
| **Protein folding (chaperones) | | **Protein folding (chaperones) |
| **Cell sorting | | **Cell sorting |
|
| |
| ==Container Specs==
| |
| [[Image:iGEM_harv06_mattspecs.gif]]
| |
|
| |
| ==Container Designs==
| |
| <gallery>
| |
| Image:Igemharv06_Katie_Val_cylinderI.gif|[[IGEM:Harvard/2006/Container Design 1|Design 1]]<br>hexagonal core, separate 1-ply lids
| |
| Image:Smallcontainerdesign2.jpg|[[IGEM:Harvard/2006/Container Design 2|Design 2]]<br>hexagonal core, separate 2-ply lids
| |
| Image:Igemharv06_msmrect.png|[[IGEM:Harvard/2006/Container Design 3|Design 3]]<br>rectangular core, continuous 1-ply lids
| |
| Image:Websmallbarrsingleply.jpg|[[IGEM:Harvard/2006/Container Design 4|Design 4]]<br>hexagonal core, separate 1-ply lids
| |
| </gallery>
| |
|
| |
| ==Latch Designs==
| |
| <gallery>
| |
| Image:iGEM_harv06_mattlatch1.jpg |latch1 <br>[[:Media:iGEM_harv06_mattlatch1.jpg|jpg]] | [[:Media:IGEM_harv06_mattlatch1.ai|ai]]
| |
| Image:iGEM_harv06_mattlatch2.jpg |latch2 <br>[[:Media:iGEM_harv06_mattlatch2.jpg|jpg]] | [[:Media:IGEM_harv06_mattlatch2.ai|ai]]
| |
| </gallery>
| |
|
| |
|
| ==Coding== | | ==Coding== |
| Line 35: |
Line 18: |
| *[[IGEM:Harvard/2006/DNA_nanostructures/Designing_DNA_nanostructures|William's code (Python)]] | | *[[IGEM:Harvard/2006/DNA_nanostructures/Designing_DNA_nanostructures|William's code (Python)]] |
|
| |
|
| ==Thrombin-aptamer experiments==
| |
|
| |
| ====Questions / procedures====
| |
| * what percent gel? 10% to 20% polyacrylamide gels, no SDS (but would make for a good control)
| |
| * what incubation conditions?
| |
| * how much protein and DNA? protein at 1 {{um}}, DNA at 2 {{um}}
| |
| * Coomassie stain
| |
|
| |
| ====Experiments====
| |
| {| {{table}}
| |
| | align="center" style="background:#f0f0f0;"|number
| |
| | align="center" style="background:#f0f0f0;"|thrombin
| |
| | align="center" style="background:#f0f0f0;"|aptamer
| |
| | align="center" style="background:#f0f0f0;"|nanotube
| |
| | align="center" style="background:#f0f0f0;"|DNA-stained prediction
| |
| | align="center" style="background:#f0f0f0;"|protein-stained prediction
| |
| |-
| |
| |0||-||-||-||no bands||no bands
| |
| |-
| |
| |1||-||-||+||slow band (nanotube)||no bands
| |
| |-
| |
| |2||-||+||-||fast band (aptamer)||no bands
| |
| |-
| |
| |3||-||+||+||slow band (aptamer-nanotube), traces of fast band (aptamer)||no bands
| |
| |-
| |
| |4||+||-||-||no bands||fast band (thrombin)
| |
| |-
| |
| |5||+||-||+||slow band (nanotube)||fast band (thrombin)
| |
| |-
| |
| |6||+||+||-||medium band (aptamer-thrombin), fast band (aptamer)||medium band (aptamer-thrombin), traces of fast band (thrombin)
| |
| |-
| |
| |7||+||+||+||very slow band (thrombin-aptamer-nanotube), slow band (aptamer-nantotube), traces of fast band (aptamer)||very slow band (thrombin-aptamer-nanotube), medium band (aptamer-thrombin), traces of fast band (thrombin)
| |
| |-
| |
| |}
| |
|
| |
| ====Buffers====
| |
| * Macaya's and Bock's selection buffer: 20 mM Tris-acetate, pH 7.4, 140 mM NaCl, 5 mM KCl, 1 mM CaCl<sub>2</sub>, 1 mM MgCl<sub>2</sub>
| |
| * Liu's incubation buffer: 40 mM Tris, 20 mM CH<sub>3</sub>COOH, 2mM EDTA, 12.5 mM (CH<sub>3</sub>COO)<sub>2</sub>Mg, pH 8.0
| |
| * Liu's PAGE buffer: 1x TAE/Mg<sup>2+</sup>
| |
|
| |
| ====Protocols====
| |
|
| |
| Potential protocol for a 2 {{ul}} incubation reaction (revised with Dr. Shih's suggestions)
| |
| * Reconsitute lyophilized [http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIAL/T6634 bovine thrombin] — ''[[User:Matthewmeisel/Notebook/2006-7-11|done]]''
| |
| * In a 0.2 mL PCR tube, mix:
| |
| ** 0.5 {{ul}} of 4x (not 5x) [[IGEM:Harvard/2006/Stock_solutions#Bock.27s_selection_buffer|Bock's selection buffer]]
| |
| ** 1.0 {{ul}} of 2 {{um}} aptamers (final concentration: 1.0 {{um}} = 2 pmol)
| |
| ** 0.5 {{ul}} of 2 {{um}} thrombin (final concentration: 0.5 {{um}} = 1 pmol)
| |
| * OR in a 0.2 mL PCR tube, mix:
| |
| ** 0.5 {{ul}} of 4x (not 5x) [[IGEM:Harvard/2006/Stock_solutions#Bock.27s_selection_buffer|Bock's selection buffer]]
| |
| ** 0.5 {{ul}} of 2 {{um}} aptamers (final concentration: 0.5 {{um}} = 1 pmol)
| |
| ** 1.0 {{ul}} of 2 {{um}} thrombin (final concentration: 1.0 {{um}} = 2 pmol)
| |
| * Alternative mix: Liu uses 10 pmol of DNA (1 {{ul}} of 10 {{um}}) and varies thrombin amount from 2 pmol (1 {{ul}} of 0.2x thrombin working stock) to 100 pmol (1 {{ul}} of 10x thrombin working stock)
| |
| * Incubate at room temperature for 30 min.
| |
| * Load onto a non-denaturing polyacrylamide gel (10% to 20% gradient) at 4{{c}}
| |
| ** Liu runs at 25 mA for 48 h.
| |
| [[User:Matthewmeisel|Matthewmeisel]] 11:11, 11 July 2006 (EDT)
| |
|
| |
| ====Bibliography====
| |
| <biblio>
| |
| # tha1 pmid=8107090
| |
| # tha2 pmid=15945116
| |
| # tha3 pmid=8298130
| |
| # tha4 pmid=1741036
| |
| # tha5 pmid=8475124
| |
| </biblio>
| |
|
| |
|
| ==Presentations== | | ==Presentations== |
