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]
- Flow anti-dig 10μl wait for 5 min.
- Flow BGB 50μl wait for 2 min.
- Flow 1.1kb (new)10μl wait for 14 min.
- Flow BGB wait none.
- Flow micro-spheres one in each chamber 10μl wait for 14 min.
- Flow BGB 50μl + seal it with nail polish
Results
1μM BEAD.[3]
{{#widget:YouTube|id=OvuG6Ju77h0}} {{#widget:YouTube|id=A_CUeRuwxco}} {{#widget:YouTube|id=ehFeJ165Tis}}
Attempt .2 (jan/11/2011)
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]
- Flow anti-dig 10μl wait for 5 min.
- Flow BGB 50μl wait for 2 min.
- Flow 1.1kb (new)10μl wait for 14 min.
- Flow BGB wait none.
- Flow micro-spheres one in each chamber 10μl wait for 14 min.
- 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.
Attempt .3 (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
Same as above.
Results
There were some tethers but no stretchable.
Attempt .5 (jan/13/2011)
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 (2:100).
Procedure
Same as above.
Results
Tethers but no stretching.
Attempt .5 (jan/19/2011)
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 (5:100).
Procedure
Same as above.
Results
Some Tethers but no stretching.
Attempt .6 (jan/20/2011)
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 (5:100).
Procedure
Same as above.
Results
Some Tethers but no stretching.
Attempt .7 (jan/24/2011)
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 (5:100).
Procedure
Same as above.
Results
Some Tethers but no significant stretching. It looked like some beads had something but nothing significant.
Attempt .8 (jan/26/2011)
components
.5μm bead with 1.1kb dna (ant just made), duplex.
- 5μl .5 μm bead + 45μl BGB =50μl (1:10).
- 1.5 μl dna + 98.5 μl POP = 100μl dna. 1.1kb dna (1:100) and (1.5:100).
Procedure
Same as above.
Results
I got possible stretching but for sure no over stretching, as bead broke off every time. [5]
Attempt .9 (jan/27/2011)
components
- Made new anti-dig (after 14 days of previous one):
180μl PBS + 20μl (anti-dig) = 200μl anti-dig. .5μm bead with 1.1kb dna (ant just made), duplex.
- 5μl .5 μm bead + 45μl BGB =50μl (1:10).
- 1.5 μl dna + 98.5 μl POP = 100μl dna. 1.1kb dna (1:100) and (1.5:100).
Procedure
Same as above.
Results
same result as the above.
Attempt .10(jan/28/2011)
components
1μm bead with 1.1kb dna (ant just made), duplex.
- 1μl .5 μm bead + 45μl BGB =50μl (1:10).
- 1 μl dna + 98.5 μl POP = 100μl dna. 1.1kb dna (1:100) and (1.5:100).
Procedure
Same as above.
Results
Tried fine tether center to work. The settings are: [6].