IGEM:Harvard/2006/Adaptamers/Notebook/2006-8-8
8/8
Experiments:
1) Speed Vac
2) thrombin beads
- 1.Speed Vac
Take remaining fraction of 8/6 elutions (theoretically 144 of 152 uL) and concentrate using the speed vac, then run on gel and stain with SYBR gold.
S0: streptavidin aptamer (40 bp single stranded) SB: streptavidin beads TB: thrombin beads
1: ladder
2: 20 pmol S0
3: 10 uL SB + 40 pmol S0
4: 25 uL SB + 40 pmol S0
5: 50 uL SB + 40 pmol S0
6: 100 uL SB + 40 pmol S0
7: 150 uL SB + 40 pmol S0
8: T35 (50bp, single stranded oligo)
9: 150uL TB + 40 pmol S0
The bands are a bit brighter, now (same imager settings). The 100uL sample has very little in it: enough beads were in this sample so that they precipitated following concentration and it was only possible to add a tiny bit of fluid.
- 2.Thrombin Beads
Repeat 8/6 experiment with thrombin beads. Slightly higher concentration of T0 will be used. Thrombin concentration (of beads) is unclear, but we'll just try different volumes.
Both gels are 4-20% polyacrylamide gels run in TBE for 50 minutes.
S0: streptavidin aptamer (40 bp single stranded) SB: streptavidin beads TB: thrombin beads
1: ladder
3: 20 pmol T0
4: 10 uL TB + 80 pmol T0
5: 25 uL TB + 80 pmol T0
6: 50 uL TB + 80 pmol T0
7: 100 uL TB + 80 pmol T0
8: 150 uL TB + 80 pmol T0
9: S5 (55bp, single stranded oligo)
10: 150uL SB + 80 pmol T0
1: ladder
2: 20 pmol T0
3: 10 uL TB + 80 pmol T0
4: 25 uL TB + 80 pmol T0
5: 50 uL TB + 80 pmol T0
6: 100 uL TB + 80 pmol T0
7: 150 uL TB + 80 pmol T0
8: S5 (55bp, single stranded oligo)
9: 150uL SB + 80 pmol T0