IGEM:Harvard/2006/Adaptamers/Notebook/2006-7-24

7/24

This time around, we're denaturing the aptamers before incubating with protein. 5 minutes at 90 degrees followed by 5 minutes at 4 degrees. Unfortunately we haven't gotten human thrombin yet. We'll also try Silver staining, which is supposed to be more sensitive than Coomassie and EtBr.

Protein

1: SeeBlue Plus 2 ladder

2: Benchmark ladder

3: thrombin (20 pmol)

4: thrombin (20 pmol) + T5 (40 pmol)

5: streptavidin (20 pmol)

6: streptavidin (20 pmol) + S5 (40 pmol)

7: streptavidin (20 pmol) + biotinylated oligos (40 pmol)

8: 1kb dna ladder

9: T5 (40 pmol)

10: T5 (40 pmol) + thrombin (20 pmol)

11: S5 (40 pmol)

12: S5 (40 pmol) + streptavidin (20 pmol)

Note: I purposely stained longer than I should because I was determined to see streptavidin. It's there, (lane 7), but it doesn't show up on its own for some reason. The large blotch in the middle of all of this is annoying, too. I'm not sure of its origin. Note for silver stainers: staining for a long time (more than 10 minutes) causes the top and bottom edges of the gels to discolor, possibly making it difficult to resolve bands.