IGEM:Harvard/2006/Adaptamers/Notebook/2006-8-1
8/1
Streptavidin beads have arrived. Product details below:
http://www.sigmaaldrich.com/catalog/search/ProductDetail/FLUKA/85881
A first experiment will just examine the effects of incubating different amounts of thrombin aptamer with the beads.
The beads are supplied at 1.2 ug/uL = 22.6 pmol/uL. Suggested amount per reaction: 25 uL or 566 pmol streptavidin. Amount of DNA added: 10 pmol, 20 pmol, 40 pmol, 80 pmol, 160 pmol, 320 pmol. Assuming capture of all DNA, if 1% of the DNA is in a solution in the last two cases, we should still be able to visualize it.
Beads were first washed 3X and transferred to equal volume of Bittker binding buffer (Bittker 2002; see [| stock solutions page]). Total bead/DNA reaction volume was 50 uL. Beads were shook for 30 minutes, then washed 3X in 25 uL Bittker buffer. Washes were collected. DNA attached to beads was eluted by shaking with 25 uL 50% wt/v streptavidin for 30 minutes followed by 25 uL Bittker buffer. 18 uL of wash and buffer solutions were then run on a 12 % polyacrylamide gel for 1 hour then stained with EtBr.
Lane 1: 1000 kb+ ladder
Lane 2: S5
Lane 5: 10 pmol S5 wash
Lane 6: 10 pmol S5 elution
Lane 7: 20 pmol S5 wash
Lane 8: 20 pmol S5 elution
Lane 9: 40 pmol S5 wash
Lane 10: 40 pmol S5 elution
Lane 11: 80 pmol S5 wash
Lane 12: 80 pmol S5 elution
Lane 1: 1000 kb+ ladder
Lane 2: S5
Lane 3: 160 pmol S5 wash
Lane 4: 160 pmol S5 elution
Lane 5: 320 pmol S5 wash
Lane 6: 320 pmol S5 elution
Lane 7: 500 pmol miniprep (nonspecific dna) wash
Lane 8: 500 pmol miniprep (nonspecific dna) elution
Lane 9: 320 pmol S5 wash (incubation with thrombin beads)
Lane 10: 320 pmol S5 elution (incubation with thrombin beads)
Several things were done wrong here:
Wash volumes were too small so lots of extra DNA was found in the elution. Incubated with miniprep dna to test non-specific DNA interaction. These didn't migrate very far. Used way too many thrombin beads. That control should have been for how much agarose S5 is binding rather than how much of a different time of protein it is binding.
Nonetheless, the miniprep DNA elution fraction seemed to have less DNA in it than elutions of other lanes with S5 and less DNA, which is somewhat promising.
