IGEM:Harvard/2006/Adaptamers/Notebook/2006-8-16

• Yesterday's gel stained with SYBR gold

Following up on yesterday's gel, we restained the gel with SYBR gold. This only caused a more diffuse signal; meanwhile T0 remained unstained.

• Yesterday's gel stained with SYBR gold one more time

• Basepairing of new adaptamers

• Streptavidin magnetic beads

We'll test the ability of our magnetic beads to bind streptavidin aptamer. The magnetic beads are able to bind ~976 ng biotin/ mL vs. 15-30 ug biotin/mL for the agarose beads. Thus we should probably use greater quantities of magnetic beads to actually see aptamer binding. At the same time, our supplies of magnetic beads are rather limited, so we'll attempt to conserve some. It will likely be possible to conserve by concentrating beads into smaller volumes.

Setup:

Magnetic beads were first washed in Bittker buffer 3X.

50, 100, or 200 uL (.4 % solid) magnetic beads concentrated to 10 uL were mixed with 40 pmol streptavidin aptamer (total of 12 uL) or 40 pmol thrombin aptamer; additionally 8 uL 10% solution was diluted to 10 uL and mixed with streptavidin aptamer as another control. Samples were then shaken for 30 minutes.

Following initial incubation, samples were washed 3X in 1 mL Bittker buffer; initial supernatant was used as wash sample in gels.

Samples were then eluted by incubating with 13 uL 1 mg/mL streptavidin.

Washes and elutions were then run on a 4-20% PA gel at 200 V for 30 minutes in TBE buffer (Novex pre-cast gel).

Lane	Condition
1	10bp ladder
2	50 uL beads sample wash
3	50 uL beads sample elution
4	100 uL beads sample wash
5	100 uL beads sample elution
6	200 uL beads sample wash
7	200 uL beads sample elution
8	200 uL beads + T35 sample wash
9	200 uL beads +T35 sample elution
10	200 uL blank beads sample wash
11	200 uL blank beads sample elution