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

7/7

Adaptamers:

We've received the short DNA sequences that we'll be using. We are waiting for purified streptavidin protein to come in. The main question is what we will use in our gel shift assay. We cannot use 32P, so our alternatives are:

Invitrogen EMSA kit (cat. E33075) $224. enough for 10 minigel assays.

Roche DIG gel shift (cat. 03353591910) $453. enough for 20 labeling rxns, 200 gel shift rxns, 20 blots, and 20 control rxns. Don't know what 'gel shift reactions' are. Also need nylon membranes, DIG wash and buffer set.

LightShift Chemiluminescent EMSA Kit (prod. # 20148) $349. Enough for 100 binding reactions (??), detection reagants for 800cm^2 membrane.

The first kit uses fluorescent dyes. We would need to buy a $600 filter for the gel imager to use it. The second two involve blotting membranes. Since this project will probably involve trying out a few types of aptamer sequences and many types of intermediate sequences between the two aptamer ends, the latter two, while supposedly more reliable, will likely be too costly. Licor also has a system based on fluorescent dyes but it looks like you buy the parts separately...

Alain suggested that all adaptamers that we create have a sequence that can bind a complementary short sequence; we'd order a lot of labeled short complementary sequence. Sounds like a good idea, as long as those short sequences do not interfere with the aptamer binding.