IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-24: Difference between revisions

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==Streptavidin binding assay==
==Streptavidin binding assay==
* For the future...
* Double asterisks indicate need for purified nanostructures
* Double asterisks indicate need for purified nanostructures
* Total volume for each reaction: 13 {{ul}}
* Total volume for each reaction: 13 {{ul}}
Line 37: Line 38:
| 12||1.0||-||-||10.0** (3.2.Lb) ||2.0||2
| 12||1.0||-||-||10.0** (3.2.Lb) ||2.0||2
|}
|}
==3.2==
====Working stocks====
Final concentration of each oligo in working stocks is 250 nM.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''working stock'''
| align="center" style="background:#f0f0f0;"|'''description'''
| align="center" style="background:#f0f0f0;"|'''pre-working stocks''' (according to [[IGEM:Harvard/2006/Container_Design_3#Working_stocks|chart]])
| align="center" style="background:#f0f0f0;"|'''water'''
| align="center" style="background:#f0f0f0;"|'''total'''
|-
|c3.2.Db||-latches<br>+aptamers_in||1 (94 {{ul}}), 2 (28 {{ul}}), 3 (30 {{ul}}), 5 (3 {{ul}}), 6b (3 {{ul}}), 8 (2 {{ul}}), 9 (0 {{ul}})||40 {{ul}}||200 {{ul}}
|-
|c3.2.Eb||-latches<br>+aptamers_out||1 (94 {{ul}}), 2 (28 {{ul}}), 3 (30 {{ul}}), 4 (3 {{ul}}), 7b (3 {{ul}}), 8 (2 {{ul}}), 9 (0 {{ul}})||40 {{ul}}||200 {{ul}}
|-
|c3.2.H.core||+latch2<br>+aptamers_in||1 (94 {{ul}}), 2 (28 {{ul}}), 3 (30 {{ul}}), 5 (3 {{ul}}), 6b (3 {{ul}}), 10 (2 {{ul}}), 12 (2 {{ul}}), 13 (0 {{ul}}), 15 (2 {{ul}})||36 {{ul}}||200 {{ul}}
|-
|c3.2.H.latches||+latch2<br>+aptamers_in||16 (4 {{ul}})||-||-
|-
|c3.2.I.core||+latch2<br>+aptamers_out||1 (94 {{ul}}), 2 (28 {{ul}}), 3 (30 {{ul}}), 4 (3 {{ul}}), 7b (3 {{ul}}), 10 (2 {{ul}}), 12 (2 {{ul}}), 13 (0 {{ul}}), 15 (2 {{ul}})||36 {{ul}}||200 {{ul}}
|-
|c3.2.I.latches||+latch2<br>+aptamers_out||16 (4 {{ul}})||-||-
|-
|c3.2.Lb||-latches<br>+aptamers_all||1 (94 {{ul}}), 2 (28 {{ul}}), 3 (30 {{ul}}), 6b (3 {{ul}}), 7b (3 {{ul}}), 8 (2 {{ul}}), 9 (0 {{ul}})||40 {{ul}}||200 {{ul}}
|}
====Folding reaction====
Reagents (each expt in respective 0.2 mL PCR tube)
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Experiment'''
| align="center" style="background:#f0f0f0;"|'''Scaffold'''
| align="center" style="background:#f0f0f0;"|'''Oligos'''
| align="center" style="background:#f0f0f0;"|'''Folding buffer'''
| align="center" style="background:#f0f0f0;"|'''d{{h2o}}'''
| align="center" style="background:#f0f0f0;"|'''total volume'''
|-
| 3.2.Db||9 {{ul}} p7308||16 {{ul}} (250 nM)||4 {{ul}} 10x||11 {{ul}}||40 {{ul}}
|-
| 3.2.Eb||9 {{ul}} p7308||16 {{ul}} (250 nM)||4 {{ul}} 10x||11 {{ul}}||40 {{ul}}
|-
| 3.2.Hb||9 {{ul}} p7308||16 {{ul}} (250 nM)||4 {{ul}} 10x||11 {{ul}}||40 {{ul}}
|-
| 3.2.Ib||9 {{ul}} p7308||16 {{ul}} (250 nM)||4 {{ul}} 10x||11 {{ul}}||40 {{ul}}
|-
| 3.2.Lb||9 {{ul}} p7308||16 {{ul}} (250 nM)||4 {{ul}} 10x||11 {{ul}}||40 {{ul}}
|-
| -oligos||9 {{ul}} p7308||-||4 {{ul}} 10x||27 {{ul}}||40 {{ul}}
|}
Annealing protocol
* start at 80{{c}}
* 60 cycles: wait 2 minutes, decrease 1{{c}}
* hold at 4{{c}}

Latest revision as of 07:56, 25 July 2006

Precipitation tests

Streptavidin binding assay

  • For the future...
  • Double asterisks indicate need for purified nanostructures
  • Total volume for each reaction: 13 μL
lane streptavidin (0.5 μM) (μL) p7308 scaffold (44 nM) (μL) oligos (250 nM) (μL) nanostructures (10 nM) (μL) water (μL) 10x Tris-gly loading dye (μL)
2 1.0 - - - 12.0 2
3 - 11.4 - - 1.6 2
4 1.0 11.4 - - 0.6 2
5 - - 4.0 (a b-working stock) - 9.0 2
6 1.0 - 4.0 (same b-working stock) - 8.0 2
7 1.0 - - 10.0** (3.2.A) 2.0 2
8 1.0 - - 10.0** (3.2.Eb or 4.0 Ib 2.0 2
9 1.0 - - 10.0** (3.2.Ib or 4.0.Ib) 2.0 2
10 1.0 - - 10.0** (3.2.Db or 4.0.Db) 2.0 2
11 1.0 - - 10.0** (3.2.Hb or 4.0.Hb) 2.0 2
12 1.0 - - 10.0** (3.2.Lb) 2.0 2

3.2

Working stocks

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

working stock description pre-working stocks (according to chart) water total
c3.2.Db -latches
+aptamers_in		 1 (94 μL), 2 (28 μL), 3 (30 μL), 5 (3 μL), 6b (3 μL), 8 (2 μL), 9 (0 μL) 40 μL 200 μL
c3.2.Eb -latches
+aptamers_out		 1 (94 μL), 2 (28 μL), 3 (30 μL), 4 (3 μL), 7b (3 μL), 8 (2 μL), 9 (0 μL) 40 μL 200 μL
c3.2.H.core +latch2
+aptamers_in		 1 (94 μL), 2 (28 μL), 3 (30 μL), 5 (3 μL), 6b (3 μL), 10 (2 μL), 12 (2 μL), 13 (0 μL), 15 (2 μL) 36 μL 200 μL
c3.2.H.latches +latch2
+aptamers_in		 16 (4 μL) - -
c3.2.I.core +latch2
+aptamers_out		 1 (94 μL), 2 (28 μL), 3 (30 μL), 4 (3 μL), 7b (3 μL), 10 (2 μL), 12 (2 μL), 13 (0 μL), 15 (2 μL) 36 μL 200 μL
c3.2.I.latches +latch2
+aptamers_out		 16 (4 μL) - -
c3.2.Lb -latches
+aptamers_all		 1 (94 μL), 2 (28 μL), 3 (30 μL), 6b (3 μL), 7b (3 μL), 8 (2 μL), 9 (0 μL) 40 μL 200 μL

Folding reaction

Reagents (each expt in respective 0.2 mL PCR tube)

Experiment Scaffold Oligos Folding buffer dH2O total volume
3.2.Db 9 μL p7308 16 μL (250 nM) 4 μL 10x 11 μL 40 μL
3.2.Eb 9 μL p7308 16 μL (250 nM) 4 μL 10x 11 μL 40 μL
3.2.Hb 9 μL p7308 16 μL (250 nM) 4 μL 10x 11 μL 40 μL
3.2.Ib 9 μL p7308 16 μL (250 nM) 4 μL 10x 11 μL 40 μL
3.2.Lb 9 μL p7308 16 μL (250 nM) 4 μL 10x 11 μL 40 μL
-oligos 9 μL p7308 - 4 μL 10x 27 μL 40 μL

Annealing protocol

  • start at 80[[:Category:{{{1}}}|{{{1}}}]]
  • 60 cycles: wait 2 minutes, decrease 1[[:Category:{{{1}}}|{{{1}}}]]
  • hold at 4[[:Category:{{{1}}}|{{{1}}}]]
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