IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-20: Difference between revisions

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==c4.0.b Folding==
===Stock oligos===
===Pre-working stocks===
*Mix 2.5 {{ul}} of each 200 {{um}} stock oligo.  2.5{{ul}} biotinylated oligos + 7.5{{uL}} water go into each pre-working stock.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''pre-working stock'''
| align="center" style="background:#f0f0f0;"|'''desc'''
| align="center" style="background:#f0f0f0;"|'''oligo tube labels'''
| align="center" style="background:#f0f0f0;"|'''total # of oligos'''
|-
|c4.0.4b||barrel oligos @ outside aptamers -aptamers +biotin||c4.0.4.1b-c4.0.4.4b||4
|-
|c4.0.6b||barrel oligos @ inside aptamers -aptamers +biotin||c4.0.6.1b-c4.0.6.4b||4
|}
===Make c4.0.b working stocks===
Final concentration of each oligo in working stocks is 250 nM. 
Working Stocks for Biotinylated Oligos
{| {{table}}
| Stock ID||Experiment||1||2||3||4||5||6||7||8||9||10||11||12||13||14||15||16||17||18||19||20||21||22||23||24||25||5b||7b
|-
| c4.0Fb||latch design 1, inside biotin||x||x||x||x||||||||x||||||x||x||||||||||||||||||||||||||||||x
|-
| c4.0Gb||latch design 1, outside biotin||x||x||x||||||x||||x||||||x||x||||||||||||||||||||||||||||x||
|-
| c4.0Hb||latch design 2, inside biotin||x||x||x||x||||||||||x||||||||x||x||||||||||||||||||x||x||x||||x
|-
| c4.0Ib||latch design 2, outside biotin||x||x||x||||||x||||||x||||||||x||x||||||||||||||||||x||x||x||x||
|}
===c4.0.b Folding Experiment===
Reagents
* 9 {{ul}} p7308 scaffold = (10 nM)/(44 nM) * 40 {{ul}}
* 16 {{ul}} oligos (3.2.A) = (100 nM)/(250 nM) * 40 {{ul}}
* 4 {{ul}} 10x folding buffer (500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM {{mgcl2}})
* 11 {{ul}} d{{h2o}}
* total volume: 40 {{ul}}
==Streptavidin-biotin binding experiments, continued==
==Streptavidin-biotin binding experiments, continued==
* goal: show that streptavidin and biotin are binding and can be imaged with PAGE
* goal: show that streptavidin and biotin are binding and can be imaged with PAGE

Revision as of 11:37, 20 July 2006

c4.0.b Folding

Stock oligos

Pre-working stocks

  • Mix 2.5 μL of each 200 μM stock oligo. 2.5μL biotinylated oligos + 7.5μL water go into each pre-working stock.
pre-working stock desc oligo tube labels total # of oligos
c4.0.4b barrel oligos @ outside aptamers -aptamers +biotin c4.0.4.1b-c4.0.4.4b 4
c4.0.6b barrel oligos @ inside aptamers -aptamers +biotin c4.0.6.1b-c4.0.6.4b 4


Make c4.0.b working stocks

Final concentration of each oligo in working stocks is 250 nM.

Working Stocks for Biotinylated Oligos

Stock ID Experiment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 5b 7b
c4.0Fb latch design 1, inside biotin x x x x x x x x
c4.0Gb latch design 1, outside biotin x x x x x x x x
c4.0Hb latch design 2, inside biotin x x x x x x x x x x x
c4.0Ib latch design 2, outside biotin x x x x x x x x x x x



c4.0.b Folding Experiment

Reagents

  • 9 μL p7308 scaffold = (10 nM)/(44 nM) * 40 μL
  • 16 μL oligos (3.2.A) = (100 nM)/(250 nM) * 40 μL
  • 4 μL 10x folding buffer (500 mM HEPES pH 7.5, 500 mM NaCl, 100 mM MgCl2)
  • 11 μL dH2O
  • total volume: 40 μL


Streptavidin-biotin binding experiments, continued

  • goal: show that streptavidin and biotin are binding and can be imaged with PAGE
    • show with both biotinylated oligos and biotinylated DNA nanostructures
lane streptavidin (2 μM) (μL) biotinylated oligo (1 μM) (μL) water (μL) loading buffer (10x Tris-glycine)
1 2 - 8 2
2 - 8 2 2
3 4 4 2 2
4 2 2 6 2
5 2 8 0 2
6 1 4 5 2
7-12 same as lanes 1-6
lane streptavidin (2 μM) (μL) box Ib (10 nM) (μL) water (μL) loading buffer (10x Tris-glycine)
1 2 - 8 2
2 - 8 2 2
3 4 4 2 2
4 2 2 6 2
5 2 8 0 2
6 1 4 5 2
7-12 same as lanes 1-6
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