IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-19: Difference between revisions
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* '''Protocol''': mix 6 {{ul}} 2 {{um}} streptavidin with 6 {{ul}} 1 {{um}} c.3.2.7.2b oligo, to give a final oligo concentration of 500 nM, and incubate at room temperature for 5 min. | * '''Protocol''': mix 6 {{ul}} 2 {{um}} streptavidin with 6 {{ul}} 1 {{um}} c.3.2.7.2b oligo, to give a final oligo concentration of 500 nM, and incubate at room temperature for 5 min. | ||
* dilution and electrophoresis: | * dilution and electrophoresis: | ||
* each lane contained 2 {{ul}} of different dilutions (as per table below), 6 {{ul}} of water, and 2 {{ul}} of loading dye | |||
** lanes 7-12 were loaded with the same mixtures as lanes 1-6, respectively | |||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''lane''' | | align="center" style="background:#f0f0f0;"|'''lane''' | ||
| align="center" style="background:#f0f0f0;"|'''volume used''' ({{ul}}) | | align="center" style="background:#f0f0f0;"|'''volume of mix used''' ({{ul}}) | ||
| align="center" style="background:#f0f0f0;"|'''dilution''' | | align="center" style="background:#f0f0f0;"|'''dilution''' | ||
| align="center" style="background:#f0f0f0;"|'''{{h2o}} added''' ({{ul}}) | |||
| align="center" style="background:#f0f0f0;"|'''final oligo concentration''' (nM) | | align="center" style="background:#f0f0f0;"|'''final oligo concentration''' (nM) | ||
| align="center" style="background:#f0f0f0;"|'''amt of oligo''' (fmols) | | align="center" style="background:#f0f0f0;"|'''amt of oligo in 2 {{ul}} diluted mix''' (fmols) | ||
|- | |- | ||
| 1||colspan=" | | 1||colspan="5"|control: 1 {{ul}} 2 {{um}} streptavidin (control) to give 2000 fmols streptavidin | ||
|- | |- | ||
| 2||colspan=" | | 2||colspan="5"|control: 2 {{ul}} 1 {{um}} oligo (control) to give 2000 fmols oligo | ||
|- | |- | ||
| 3||2||1x||500||1000 | | 3||2||1x||0||500||1000 | ||
|- | |- | ||
| 4|| | | 4||1||5x||4||100||250 | ||
|- | |- | ||
| 5|| | | 5||1||10x||9||50||100 | ||
|- | |- | ||
| 6|| | | 6||1||40x||39||12.5||25 | ||
|} | |} | ||
* ran on native polyacrylamide gel for 30 min. at 120V | * ran on native polyacrylamide gel for 30 min. at 120V | ||
Revision as of 08:36, 19 July 2006
Protection assay
Incubation and dilution protocol, take 2
- Goal: to see the lower threshold of imaging streptavidin bound to biotin
- and to see if we can image any streptavidin-biotin construct on a gel at all
- Protocol: mix 6 μL 2 μM streptavidin with 6 μL 1 μM c.3.2.7.2b oligo, to give a final oligo concentration of 500 nM, and incubate at room temperature for 5 min.
- dilution and electrophoresis:
- each lane contained 2 μL of different dilutions (as per table below), 6 μL of water, and 2 μL of loading dye
- lanes 7-12 were loaded with the same mixtures as lanes 1-6, respectively
| lane | volume of mix used (μL) | dilution | H2O added (μL) | final oligo concentration (nM) | amt of oligo in 2 μL diluted mix (fmols) |
| 1 | control: 1 μL 2 μM streptavidin (control) to give 2000 fmols streptavidin | ||||
| 2 | control: 2 μL 1 μM oligo (control) to give 2000 fmols oligo | ||||
| 3 | 2 | 1x | 0 | 500 | 1000 |
| 4 | 1 | 5x | 4 | 100 | 250 |
| 5 | 1 | 10x | 9 | 50 | 100 |
| 6 | 1 | 40x | 39 | 12.5 | 25 |
- ran on native polyacrylamide gel for 30 min. at 120V
