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

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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

Cst808concelution.jpg

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

Cst808thrombelution.jpg

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

Cst808thrombwash.jpg