IGEM:Harvard/2006/Adaptamers/Notebook/2006-7-11
7/11
Shawn and William suggested that for an initial assay, it'd be easy to just stain protein with Coomassie blue. So that's what we attempted.
We ran two 10-20% native polyacrylamide gels at 15 V overnight; the first used some lanes from the Nano group's gel. Visualization to come tomorrow.
The composition of what we added to each line is below; materials were added and incubated for 30 minutes in 2 uL total volume for lanes 1-11, 1-12 prior to dilution to 10 uL including addition of loading dye. Lanes 2-1 to 2-9 were incubated for an hour; longer incubation carried out since DNA basepairing reactions for lanes 2-10 to 2-12 were carried out for 30 minutes using results from 30-minute incubations of lanes 2-1 to 2-9. This incubation time should not be significant.
Materials used: Thrombin: a 2 uM solution of Thrombin. Streptavidin: a 2 uM solution of Streptavidin. Thromb5, Thromb20, Thromb35, Thromb50, Strep5, Strep20, Strep35, Strep50 are described in Plan section above. Bock's buffer is a reaction buffer for DNA-protein binding, used in several papers (see Nano group's page). 10X dye is a few flakes of bromophenol blue + 500 uL Bock's + 500 uL glycerol.
All quantities in uL.
| 1-11 | 1-12 | 2-1 | 2-2 | 2-3 | 2-4 | 2-5 | 2-6 | 2-7 | 2-8 | 2-9 | 2-10 | 2-11 | 2-12 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Thrombin | .5 | .5 | .5 | 1 | 1 | 1 | ||||||||
| Streptavidin | .5 | .5 | 1 | 1 | 1 | |||||||||
| Thromb5 | 1 | 1 | ||||||||||||
| Thromb20 | 2 | |||||||||||||
| Thromb35 | 2 | |||||||||||||
| Thromb50 | 2 | |||||||||||||
| Strep5 | 2 | |||||||||||||
| Strep20 | 2 | |||||||||||||
| Strep35 | 2 | |||||||||||||
| Strep50 | 2 | |||||||||||||
| 4X Bock's Buffer (see Nanostructure page) | .5 | .5 | .5 | 1 | 1 | 1 | .5 | .5 | 1 | 1 | 1 | |||
| H2O | 1 | 1 | ||||||||||||
| 2-2 solution | 2 | |||||||||||||
| 2-3 solution | 2 | |||||||||||||
| 2-4 solution | 2 | |||||||||||||
| 2-7 | 2 | |||||||||||||
| 2-8 | 2 | |||||||||||||
| 2-9 | 2 | |||||||||||||
| Bock's 1X buffer | 7 | 7 | 7 | 7 | 7 | 7 | 7 | 7 | 7 | 7 | 7 | 5 | 5 | 5 |
| 10X dye | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Ran 2 10-20% polyacrylamide gels along with nano group.
