User:Pranav Rathi/Notebook/OT/2011/01/10/DNA tethering experiments

Atempt .1 (jan/10/2011)

I attempt tethering again today with new beads and new anti-dig.

Ant-dig

Dr. Koch and I prepared new anti-dig: 1X concentration

• PBS [1]180μl + aliquots 20μl = [MIX] = Anti-dig 200μl.

PBS was used from the glass door fridge and aliquot from super cold gray fridge. I used two different beads:

• 1 μm new beads (looks good in microscope), 20μl from the batch. 5μl (batch) +4 5μl BGB = 50μl.
• .5μm (FL) expired on 01/07/2010.5μl (batch)+45μl BGB = 50μl.

Procedure[2]

1. Flow anti-dig 10μl wait for 5 min.
2. Flow BGB 50μl wait for 2 min.
3. Flow 1.1kb (new)10μl wait for 14 min.
4. Flow BGB wait none.
5. Flow micro-spheres one in each chamber 10μl wait for 14 min.
6. Flow BGB 50μl + seal it with nail polish

Results

1μM BEAD.[3]

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Atempt .2 (jan/12/2011)

components

.5μm bead with 1.1kb dna (ant just made), duplex.

• 5μl .5 μm bead + 45μl BGB =50μl (1:10).
• 1.1kb dna (1:100) and (1.5:100).

Procedure[4]

1. Flow anti-dig 10μl wait for 5 min.
2. Flow BGB 50μl wait for 2 min.
3. Flow 1.1kb (new)10μl wait for 14 min.
4. Flow BGB wait none.
5. Flow micro-spheres one in each chamber 10μl wait for 14 min.
6. Flow BGB 50μl + seal it with nail polish

Results

Both the chambers were same no difference between 1 and 1.5 to 100 concentration. Hardly any tethers. The one found, break immediately.