User:Anthony Salvagno/Notebook/Research/2009/10/15/Making Tethers

Supplies

• I made 1x popping buffer
• I made k-casein in 1x pop (5mg/ml) hopefully this is a good blocker
• Need a-dig
• DNA
• beads

A-dig

For this there are 20ul aliquots and I need 1:10 of that so add 180ul 1x Pop

DNA

I can try and tether both strectching DNA 4.4 kb and some unzipping stuff. I should dilute about 1:10 fold for both so that I can see if I got anything. I think we went more dilute, but I want tethers.

Beads

I need to dilute beads (amount TBD after looking through notebook), then sonicate to remove clumps. Following this protocol: User:Anthony Salvagno/Notebook/Research/2009/08/06/Bead Washing

OK Beads are now made. I don't think sonicating worked well.

Tethering

Ok, now everything is ready. It is time to tether.

1. Flow a-dig
   • allow to sit 5min
2. flow BGB
   • allow to sit 2min
3. flow DNA
   • flowing 4.4kb stretch DNA and TD(1) unzipping DNA
   • allow to sit 5min
4. wash with Pop
5. flow Spheres
   • allow to sit 7min
6. wash with pop
7. Seal with nail polish